N-Aryl Pyrrolidine Derivatives as Beta-Secretase Inhibitors

ABSTRACT

There is provided a series of substituted N-aryl pyrrolidine derivatives of Formula (I) 
     
       
         
         
             
             
         
       
     
     or a stereoisomer; or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 5′ , R 6 , R 7 , and p as defined herein, their pharmaceutical compositions and methods of use. These compounds inhibit the processing of amyloid precursor protein (APP) by β-secretase and, more specifically, inhibit the production of Aβ-peptide. The present disclosure is directed to compounds useful in the treatment of neurological disorders related to β-amyloid production, such as Alzheimer&#39;s disease and other conditions affected by anti-amyloid activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a non-provisional application which claims the benefit of U.S. Provisional Application No. 60/788,844 filed Apr. 3, 2006.

FIELD OF THE DISCLOSURE

This patent application provides N-aryl pyrrolidine derivatives having drug and bio-affecting properties, their pharmaceutical compositions and method of use. In particular, the disclosure is concerned with a series of N-aryl pyrrolidine derivatives which are inhibitors of β-amyloid peptide (β-AP) production, thereby acting to prevent the accumulation of amyloid protein deposits in the brain and, therefore, are useful in the treatment of neurological disorders related to β-amyloid production. More particularly, the present disclosure relates to the treatment of Alzheimer's Disease (AD) and similar diseases.

BACKGROUND

Alzheimer's Disease is a progressive, neurodegenerative disorder characterized by memory impairment and cognitive dysfunction. AD is characterized pathologically by the accumulation of senile (neuritic) plaques, neurofibrillary tangles, amyloid deposition in neural tissues and vessels, synaptic loss, and neuronal death. It is the most common form of dementia and it now represents the third leading cause of death after cardiovascular disorders and cancer. The cost of Alzheimer's Disease is enormous (in the U.S., greater than $100 billion annually) and includes the suffering of the patients, the suffering of families, and the lost productivity of patients and caregivers. As the longevity of society increases, the occurrence of AD will markedly increase. It is estimated that more than 10 million Americans will suffer from AD by the year 2020, if methods for prevention and treatment are not found. Currently, AD is estimated to afflict 10% of the population over age 65 and up to 50% of those over the age of 85. No treatment that effectively prevents AD or reverses the clinical symptoms and underlying pathophysiology is currently available (for review, see Selkoe, D. J. Ann. Rev. Cell Biol., 1994, 10: 373-403).

Histopathological examination of brain tissue derived upon autopsy or from neurosurgical specimens in affected individuals reveals the occurrence of amyloid plaques and neurofibrillar tangles in the cerebral cortex of such patients. Similar alterations are observed in patients with Trisomy 21 (Down's syndrome). Biochemical and immunological studies reveal that the dominant proteinaceous component of the amyloid plaque is an approximately 4.2 kilodalton (kD) protein of about 39 to 43 amino acids. This protein is designated Aβ, β-amyloid peptide, and sometimes β/A4; referred to herein as Aβ. In addition to its deposition in amyloid plaques, Aβ is also found in the walls of meningeal and parenchymal arterioles, small arteries, capillaries, and sometimes, venules. Compelling evidence accumulated during the last decade reveals that Aβ is an internal polypeptide derived from a type 1 integral membrane protein, termed β-amyloid precursor protein (APP) (Selkoe, D. Physiol. Rev. 2001, 81, 741-766; Wolfe, M. J. Med. Chem. 2001, 44, 2039-2060). βAPP is normally produced by many cells both in vivo and in cultured cells, derived from various animals and humans. Several proteolytic fragments of APP are generated by proteinases referred to as secretases. A subset of these proteolytic fragments, designated β-amyloid peptide (Aβ), contains 39 to 43 amino acids and is generated by the combined action of β-secretase and γ-secretase. β-secretase is a membrane-bound, aspartyl protease that forms the N-terminus of the Aβ peptide. The C-terminus of the Aβ peptide is formed by γ-secretase, an apparently oligomeric complex that includes presenilin-1 and/or presenilin-2. Presenilin-1 and presenilin-2 are polytopic membrane-spanning proteins that may contain the catalytic components of γ-secretase (Seiffert, D.; Bradley, J. et al., J. Biol. Chem. 2000, 275, 34086-34091).

In addition to AD, excess production and/or reduced clearance of Aβ causes cerebral amyloid angiopathy (CAA) (reviewed in Thal, D., Gherbremedhin, E. et al., J. Neuropath. Exp. Neuro. 2002, 61, 282-293). In these patients, vascular amyloid deposits cause degeneration of vessel walls and aneurysms that may be responsible for 10-15% hemorrhagic strokes in elderly patients. As in AD, mutations in the gene encoding Aβ lead to an early onset form of CAA, referred to as cerebral hemorrhage with amyloidosis of the Dutch type, and mice expressing this mutant protein develop CAA that is similar to patients.

A logical approach to reducing Aβ levels is to interfere with the action of the secretases that are directly involved in the cleavage of APP to Aβ. The β-secretase enzyme (BACE) is responsible for cleaving APP and forms the amino-terminus of Aβ, initiating the amyloidogenic pathway. The BACE enzyme is a transmembrane aspartyl protease and was described in the literature by several independent groups [see Hussain, I. et al., (1999) Mol. Cell. Neurosci., 14: 419-427; Lin, X. et al., (2000) Proceedings of the National Academy of Sciences of the United States of America, 97: 1456-1460; Sinha, S., et al., (1999) Nature (London), 402: 537-540; Vassar, R., et al., (1999) Science (Washington, D.C.), 286: 735-741; Walsh, D. M. et al., (2002); Wolfe, M. S. (2001); Yan, R. et al., (1999) Nature (London), 402: 533-537].

Removal of BACE activity in mice by gene targeting completely abolishes Aβ production [see Luo, Y., et al., (2001) Nature Neuroscience, 4: 231-232; Roberds, S. L. et al., (2001) Human Molecular Genetics, 10: 1317-1324].

BACE −/− mice also show no detectable negative phenotypes, suggesting that disruption of BACE-mediated cleavage of APP does not produce additional undesired effects. This demonstrates that a drug substance capable of inhibiting β-secretase activity should lower or halt the synthesis of Aβ and should provide a safe treatment for Alzheimer's disease.

At present there remains an urgent need to develop pharmaceutical agents capable for effective treatment in halting, slowing, preventing, and/or reversing the progression of Alzheimer's disease. Compounds that are effective inhibitors of beta-secretase, that inhibit beta-secretase mediated cleavage of APP, that are effective inhibitors of Aβ protein production by beta-secretase, and/or are effective in reducing soluble Aβ protein, amyloid beta deposits or amyloid beta plaques, are needed for effective treatment in halting, slowing, preventing, and/or reversing neurological disorders related to Aβ protein production, such as Alzheimer's disease.

SUMMARY OF THE DISCLOSURE

A series of N-aryl pyrrolidine derivatives having the Formula (I)

or a stereoisomer; or a pharmaceutically acceptable salt thereof, wherein R₁, R₂, R₃, R₄, R₅, R_(5′), R₆, R₇ and p as defined below are effective inhibitors of the production of β-amyloid peptide (β-AP) from β-amyloid precursor protein (β-APP). The pharmacologic action of these compounds makes them useful for treating conditions responsive to the inhibition of β-AP in a patient; e.g., Alzheimer's Disease (AD) and Down's Syndrome. Therapy utilizing administration of these compounds or a pharmaceutical composition containing a therapeutically effective amount of at least one of these compounds to patients suffering from, or susceptible to, these conditions involves reducing β-AP available for accumulation and deposition in brains of these patients.

DETAILED DESCRIPTION

The present application comprises compounds of Formula I, their pharmaceutical formulations, and their use in inhibiting β-AP production in patients suffering from or susceptible to AD or other disorders resulting from β-AP accumulation in brain tissue. The compounds of Formula I which include stereoisomers and pharmaceutically acceptable salts thereof have the following formula and meanings:

wherein

-   R₁ is phenyl optionally substituted with one or more groups selected     from halogen, CN, CF₃, OH, —NH₂, C₃₋₆cycloalkyl, C₁₋₆alkoxy,     C₂₋₆alkenyl, C₁₋₆alkyl optionally substituted with OH,     C₃₋₆cycloalkyl, or —NH₂, —(CH₂)_(m)—NHC(═O)OC₁₋₆ alkyl,     —(CH₂)_(m)—NHC(═O)Ophenyl optionally substituted with halogen,     —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉; -   R₂ and R₃ are each independently hydrogen, methyl or hydroxymethyl; -   p is 0 or 1; -   m is 0 or 1; -   R₄ and R₆ are independently hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH,     NH₂, benzyloxy, phenyl or CF₃ in which said phenyl is optionally     substituted with one or more groups selected from halogen, CN, CF₃     and OCH₃; -   R₅ and R₅ are each independently hydrogen, C₁₋₆alkyl, or     C₁₋₄alkanol; -   R₇ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂,     N(CH₃)₂, NO₂, or CF₃; -   R₈ is C₁₋₆alkyl or C₃₋₆cycloalkyl in which each is optionally     substituted with a group selected from halogen, CN, CF₃ and     C₁₋₄alkoxy; -   R₉ is —C₁₋₆alkylNR₁₀R₁₁; -   R₁₀ is hydrogen or C₁₋₆alkyl; and -   R₁₁ is hydrogen, C₁₋₆alkyl optionally substituted with OH, halogen,     C₁₋₄alkoxy, C₃₋₆cycloalkyl, thienyl or C₁₋₄alkylcarboxyl,     —(CH₂)_(m)C₃₋₆cycloalkyl optionally substituted with phenyl or     C₁₋₄alkyl; —(CH₂)_(m)phenyl optionally substituted with halogen,     hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy; or R₁₀ and R₁₁ together with the     nitrogen to which they are attached is azetidine, aziridine,     pyrrolidine, piperidine, homopiperidine, homopiperazine, morpholine     or thiomorpholine, in which each is optionally substituted with a     group selected from halogen, C₁₋₆alkyl and C₁₋₄alkoxy;     or a nontoxic pharmaceutically acceptable salt thereof.

The present application also provides a method for the treatment or alleviation of disorders associated with β-amyloid peptide, especially Alzheimer's Disease, cerebral amyloid angiopathy and Down's Syndrome, which comprises administering together with a conventional adjuvant, carrier or diluent a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

As used herein, the term “Aβ” denotes the protein designated Aβ, β-amyloid peptide, and sometimes β/A4, in the art. Aβ is an approximately 4.2 kilodalton (kD) protein of about 39 to 43 amino acids found in amyloid plaques, the walls of meningeal and parenchymal arterioles, small arteries, capillaries, and sometimes, venules. The isolation and sequence data for the first 28 amino acids are described in U.S. Pat. No. 4,666,829. The 43 amino acid sequence is well known in the art, see Colin Dingwall, Journal of Clinical Investigation, November 2001, 108 (9): 1243-1246; as well as PCT international patent application WO 01/92235, published Dec. 6, 2001, herein incorporated by reference in its entirety.

The term “APP”, as used herein, refers to the protein known in the art as β amyloid precursor protein. This protein is the precursor for Aβ and through the activity of “secretase” enzymes, as used herein, it is processed into Aβ. Differing secretase enzymes, known in the art, have been designated 0 secretase, generating the N-terminus of Aβ, α secretase cleaving around the 16/17 peptide bond in Aβ, and “γ secretases”, as used herein, generating C-terminal Aβ fragments ending at position 38, 39, 40, 42, and 43 or generating C-terminal extended precursors which are subsequently truncated to the above polypeptides.

The term “substituted,” as used herein and in the claims, means that any one or more hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound.

As used herein and in the claims, “alkyl” or “alkylene” is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms; for example, “C₁₋₆ alkyl” denotes alkyl having 1 to 6 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, pentyl, 3-methylbutyl, hexyl and the like. Preferred “alkyl” group, unless otherwise specified, is “C₁₋₄ alkyl”. Additionally, unless otherwise specified, “propyl” denotes n-propyl or i-propyl; “butyl” denotes n-butyl, i-butyl, sec-butyl, or t-butyl.

As used herein and in the claims, “alkanol” is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups bearing a hydroxyl group having the specified number of carbon atoms; for example, “C₁₋₆ alkanol” denotes alkanol having 1 to 6 carbon atoms. Examples of alkyl include, but are not limited to, hydroxymethyl, hydroxyethyl, 3-hydroxy-n-propyl, 2-hydroxy-i-propyl, 4-hydroxy-n-butyl, and the like. Preferred “alkanol” group, unless otherwise specified, is “C₁₋₄ alkanol”.

As used herein and in the claims, “alkenyl” or “alkenylene” is intended to include hydrocarbon chains of either a straight or branched configuration and one or more unsaturated carbon-carbon bonds which may occur in any stable point along the chain, for example, “C₂₋₆ alkenyl” include but are not limited to ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 3-methyl-2-butenyl, 2-pentenyl, 3-pentenyl, hexenyl, and the like.

As used herein and in the claims, “alkynyl” or “alkynylene” is intended to include hydrocarbon chains of either a straight or branched configuration and one or more carbon-carbon triple bonds which may occur in any stable point along the chain, for example, “C₂₋₆ alkynyl” include but not limited to ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, and the like.

“Alkoxy” or “alkyloxy” represents an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. Examples of “C₁₋₆alkoxy” include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, and s-pentoxy. Preferred alkoxy groups are methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy.

As used herein and in the claims, “halogen” refers to fluoro, chloro, bromo, and iodo. Unless otherwise specified, preferred halogens are fluoro and chloro. “Counterion” is used to represent a small, negatively charged species such as chloride, bromide, hydroxide, acetate, sulfate, and the like.

“Cycloalkyl” is intended to include saturated ring groups, having the specified number of carbon atoms. For example, “C₃₋₆ cycloalkyl” denotes such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The compounds described herein may have asymmetric centers. It is understood, that whether a chiral center in an isomer is “R” or “S” depends on the chemical nature of the substituents of the chiral center. All configurations of compounds of the invention are considered part of the invention. Additionally, the carbon atom to which R₂ and R₃ is attached and carbon atoms of the pyrrolidine ring may describe a chiral carbon. Compounds of the present disclosure containing an asymmetrically substituted atom may be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials. Mixtures of isomers of the compounds of the examples or chiral precursors thereof can be separated into individual isomers according to methods which are known per se, e.g. fractional crystallization, adsorption chromatography or other suitable separation processes. Resulting racemates can be separated into antipodes in the usual manner after introduction of suitable salt-forming groupings, e.g. by forming a mixture of diastereosiomeric salts with optically active salt-forming agents, separating the mixture into diastereomeric salts and converting the separated salts into the free compounds. The enantiomeric forms may also be separated by fractionation through chiral high pressure liquid chromatography columns. Many geometric isomers of olefins and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms. All chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein and in the claims, “pharmaceutically acceptable salts” refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.

The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, the disclosure of which is hereby incorporated by reference.

In the method of the present application, the term “therapeutically effective amount” means the total amount of each active component of the method that is sufficient to show a meaningful patient benefit, i.e., healing of acute conditions characterized by inhibition of β-amyloid peptide production. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. The terms “treat, treating, treatment” as used herein and in the claims means preventing or ameliorating diseases associated with β-amyloid peptide.

The compounds of the present application can be prepared in a number of ways well known to one skilled in the art of organic synthesis. The compounds of the present application can be synthesized using the methods described below, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art. Preferred methods include, but are not limited to, those described below. All references cited herein are hereby incorporated in their entirety herein by reference.

The compounds may be prepared using the reactions and techniques described in this section. The reactions are performed in solvents appropriate to the reagents and materials employed and are suitable for the transformations being effected. Also, in the description of the synthetic methods described below, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, are chosen to be the conditions standard for that reaction, which should be readily recognized by one skilled in the art. It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the molecule must be compatible with the reagents and reactions proposed. Such restrictions to the substituents which are compatible with the reaction conditions will be readily apparent to one skilled in the art and alternate methods must then be used.

Compounds of formula I are prepared as illustrated in Scheme 1. Treatment of the appropriate pyrrolidine derivative (II) with an aryl halide (bromide or iodide are preferred) in the presence of CuO at elevated temperatures affords the N-aryl intermediates of formula III. Amide bond coupling of III with N—Boc-S-Methylisothiourea affords compounds IV. Intermediates of formula IV are then combined with amines V in the presence of organic base to afford the penultimate compounds VI. Intermediates of formula V can be obtained commercially, can be prepared by methods known in the literature, or can be readily prepared by one skilled in the art. Treatment of intermediates of formula VI with trifluoroacetic acid (TFA) provides compounds of formula Ia.

Compounds of formula II are generally known in the literature and a large number of these derivatives are available to one skilled in the art. Representative procedures for forming substituted pyrrolidines can be found in many reviews, including Coldham, I. and Hufron, R. “Intramolecular dipolar cycloaddition reactions of azomethine ylides” Chemical Reviews, 2005, 105 (7), 2765-2809.

A particularly useful reaction in this regard is the 2+3 cycloaddition of azomethine ylides. One variant of this reaction is shown in Scheme 2 (Philip Garner and Uemit Kaniskan, Tetrahderon Letters, 2005, 46 (31), 5181), and there are many additional types known in the literature and accessible to one skilled in the art.

Additionally, compounds of formula Ib wherein R represents hydrogen or one or more substituents selected from the groups defined herein are prepared by further modifying intermediates of formula VIa where an aniline functionality is present. For instance (Scheme 3), intermediate VIa is sequentially treated with bromoacetyl bromide to form the bromomethyl amide, then with an appropriate amine to afford the intermediate VII. Appropriate amines can be obtained commercially, can be prepared by methods known in the literature, or can be readily prepared by one skilled in the art. Deprotection with TFA then affords compounds Ib.

Compounds of formula Va are prepared from intermediates of formula VIII as outlined in Scheme 4. Treatment of intermediates of formula VIII with N-bromosuccinimide in the presence of a radical initiator (AIBN or benzoylperoxide) provides intermediates of formula IX. Intermediates of formula VIII can be obtained commercially, can be prepared by methods known in the literature, or can be readily prepared by one skilled in the art. Intermediates of formula IX are converted to compounds of formula Va via two routes. In the first route, intermediates of formula IX are treated with sodium azide to provide intermediates of formula X, which upon treatment with lithium aluminum hydride affords compounds of formula Va. Alternatively, intermediates of formula IX are treated with the potassium salt of phthalimide to provide intermediates of formula XI, which upon treatment with hydrazine affords compounds of formula Va.

Compounds of formula Vb are also prepared from nitrile-containing intermediates of formula XII as outlined in Scheme 5. Treatment of intermediates of formula XII with strong hydride-based reducing agents such as lithium aluminum hydride or borane provided compounds of formula Vb.

Compounds of formula Vb are also prepared from carboxylic acid derivatives of formula XIII as outlined in Scheme 6. Treatment of intermediates of formula XIII with ammonia under typical amide bond formation conditions affords intermediates of formula XIV, which upon treatment with strong hydride-based reducing agents such as borane provides compounds of formula Vb.

In a preferred, the present invention includes compounds of Formula (II) or a stereoisomer thereof

wherein R₁ is phenyl optionally substituted with one or more groups selected from halogen, CN, CF₃, OH, —NH₂, C₃₋₆cycloalkyl, C₁₋₆alkoxy, C₂₋₆alkenyl, C₁₋₆alkyl optionally substituted with OH, C₃₋₆cycloalkyl, or —NH₂, —(CH₂)_(m)—NHC(═O)OC₁₋₆ alkyl, —(CH₂)_(m)—NHC(═O)Ophenyl optionally substituted with halogen, —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉; p is 0 or 1; m is 0 or 1; R₄ is hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy, phenyl or CF₃ in which said phenyl is optionally substituted with one or more groups selected from halogen, CN, CF₃ and OCH₃; R₇ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ is C₁₋₆alkyl or C₃₋₆cycloalkyl in which each is optionally substituted with a group selected from halogen, CN, CF₃ and C₁₋₄alkoxy; R₉ is —C₁₋₆alkylNR₁₀R₁₁; R₁₀ is hydrogen or C₁₋₆alkyl; and R₁₁ is hydrogen, C₁₋₆alkyl optionally substituted with OH, halogen, C₁₋₄alkoxy, C₃₋₆cycloalkyl, thienyl or C₁₋₄alkylcarboxyl, —(CH₂)_(m)C₃₋₆cycloalkyl optionally substituted with phenyl or C₁₋₄alkyl; —(CH₂)_(m)phenyl optionally substituted with halogen, hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy; or R₁₀ and R₁₁ together with the nitrogen to which they are attached is azetidine, aziridine, pyrrolidine, piperidine, homopiperidine, homopiperazine, morpholine or thiomorpholine, in which each is optionally substituted with a group selected from halogen, C₁₋₆alkyl and C₁₋₄alkoxy; or a nontoxic pharmaceutically acceptable salt thereof.

In another preferred embodiment, the present invention includes compounds of Formula (III) or a stereoisomer thereof

wherein R₁ is phenyl optionally substituted with one or more groups selected from halogen, C₃₋₆cycloalkyl, C₁₋₆alkyl optionally substituted with cyclopropyl, or —(CH₂)_(m)—, —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉; R₄ is hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy, phenyl or CF₃ in which said phenyl is optionally substituted with one or more groups selected from halogen, CN, CF₃ and OCH₃; R₇ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ is C₁₋₆alkyl or C₃₋₆cycloalkyl in which each is optionally substituted with a group selected from halogen, CN, CF₃ and C₁₋₄alkoxy; R₉ is —C₁₋₆alkylNR₁₀R₁₁; R₁₀ is hydrogen or C₁₋₆alkyl; and R₁₁ is hydrogen, C₁₋₆alkyl optionally substituted with OH, halogen, C₁₋₄alkoxy, C₃₋₆cycloalkyl, thienyl or C₁₋₄alkylcarboxyl, —(CH₂)_(m)C₃₋₆cycloalkyl optionally substituted with phenyl or C₁₋₄alkyl; —(CH₂)_(m)phenyl optionally substituted with halogen, hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy; or R₁₀ and R₁₁ together with the nitrogen to which they are attached is azetidine, aziridine, pyrrolidine, piperidine, homopiperidine, homopiperazine, morpholine or thiomorpholine, in which each is optionally substituted with a group selected from halogen, C₁₋₆alkyl and C₁₋₄alkoxy; m is 0 or 1; or a nontoxic pharmaceutically acceptable salt thereof.

In still another preferred embodiment, the present invention includes compounds of Formula (IV)

wherein R₁ is phenyl optionally substituted with one or more groups selected from halogen, C₃₋₆cycloalkyl, C₁₋₆alkyl optionally substituted with cyclopropyl, or —(CH₂)_(m)—, —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉; R₄ is hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy, phenyl or CF₃ in which said phenyl is optionally substituted with one or more groups selected from halogen, CN, CF₃ and OCH₃; R₇ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ is C₁₋₆alkyl or C₃₋₆cycloalkyl in which each is optionally substituted with a group selected from halogen, CN, CF₃ and C₁₋₄alkoxy; R₉ is —C₁₋₆alkylNR₁₀R₁₁; R₁₀ is hydrogen or C₁₋₆alkyl; and R₁₁ is hydrogen, C₁₋₆alkyl optionally substituted with OH, halogen, C₁₋₄alkoxy, C₃₋₆cycloalkyl, thienyl or C₁₋₄alkylcarboxyl, —(CH₂)_(m)C₃₋₆cycloalkyl optionally substituted with phenyl or C₁₋₄alkyl; —(CH₂)_(m)phenyl optionally substituted with halogen, hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy; or R₁₀ and R₁₁ together with the nitrogen to which they are attached is azetidine, aziridine, pyrrolidine, piperidine, homopiperidine, homopiperazine, morpholine or thiomorpholine, in which each is optionally substituted with a group selected from halogen, C₁₋₆alkyl and C₁₋₄alkoxy; m is 0 or 1; or a nontoxic pharmaceutically acceptable salt thereof.

In yet another preferred embodiment, the present invention includes compounds of Formula (IV)

wherein R₁ is phenyl optionally substituted with one or more groups selected from halogen, C₃₋₆cycloalkyl, C₁₋₆alkyl optionally substituted with cyclopropyl, or —(CH₂)_(m)—, —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉; R₄ is hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, benzyloxy, phenyl or CF₃ in which said phenyl is optionally substituted with one or more groups selected from halogen, CN, CF₃ and OCH₃; R₇ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ is C₁₋₆alkyl or C₃₋₆cycloalkyl in which each is optionally substituted with a group selected from halogen, CN, CF₃ and C₁₋₄alkoxy; R₉ is —C₁₋₆alkylNR₁₀R₁₁; R₁₀ is hydrogen or C₁₋₆alkyl; and R₁₁ is hydrogen, C₁₋₆alkyl optionally substituted with OH, halogen, C₁₋₄alkoxy, C₃₋₆cycloalkyl, —(CH₂)_(m)C₃₋₆cycloalkyl; —(CH₂)_(m)phenyl optionally substituted with halogen, hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy; or R₁₀ and R₁₁ together with the nitrogen to which they are attached is azetidine, aziridine, pyrrolidine, piperidine, homopiperidine, homopiperazine, morpholine or thiomorpholine; m is 0 or 1; or a nontoxic pharmaceutically acceptable salt thereof.

In a further embodiment, this invention includes pharmaceutical compositions comprising at least one compound of Formula I in combination with a pharmaceutical adjuvant, carrier or diluent.

In another further embodiment, this invention relates to a method of treatment or prevention of disorders responsive to the inhibition of β-amyloid peptide in a mammal in need thereof, which comprises administering to said mammal a therapeutically effective amount of a compound of Formula I or a nontoxic pharmaceutically acceptable salt.

In yet another further embodiment, this invention relates to a method for treating Alzheimer's Disease, cerebral amyloid angiopathy and Down's Syndrome in a mammal in need thereof, which comprises administering to said mammal a therapeutically effective amount of a compound of Formula I or a non-toxic pharmaceutically acceptable salt.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The compounds of this application and their preparation can be understood further by the following working examples. These examples are meant to be illustrative of the present application, and are not to be taken as limiting thereof.

Chemical abbreviations used in the specification and Examples are defined as follows:

-   “Ac” for acetate, -   “APCI” for atmospheric pressure chemical ionization, -   “Boc” or “BOC” for t-butyloxycarbonyl, -   “BOP” for benzotriazol-1-yloxytris-(dimethylamino)-phosphonium     hexafluorophosphate, -   “Cbz” for benzyloxycarbonyl, -   “CDI” for 1,1′-carbonyldiimidazole, -   “CD₃OD” for deuteromethanol, -   “CDCl₃” for deuterochloroform, -   “DCC” for 1,3-dicyclohexylcarbodiimide, -   “DCM” for dichloromethane -   “DEAD” for diethyl azodicarboxylate, -   “DIEA”, “Hunig's base”, or “DIPEA” for N,N-diisopropylethylamine, -   “DMF” for N,N-dimethylformamide, -   “DMAP” for 4-dimethylaminopyridine, -   “DMPU” for 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidone, -   “DMSO” for dimethylsulfoxide, -   “DPPA” for diphenylphosphorylazide -   “EDC” or “EDCI” for 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide     hydrochloride, -   “Et” for ethyl, -   “EtOAC” for ethyl acetate, -   “HOAc” for acetic acid, -   “HOBt” for 1-hydroxybenzotriazole hydrate, -   “HATU” for O-(7-azabenzotriazol-1-yl)-N,N, N′,N′-tetramethyluronium     hexafluorophosphate, -   “HMPA” for hexamethylphosphoramide, -   “LDA” for lithium diisopropylamide, -   “LiHMDS” for lithium bis(trimethylsilyl)amide, -   “NaHMDS” for sodium bis(trimethylsilyl)amide, -   “NMM” for 4-methylmorpholine, -   “PyBOP” for benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium     hexafluorophosphate, -   “TMSCH₂N₂” for (trimethylsilyl)diazomethane, -   “TMSN₃” for Azidotrimethylsilane, -   “TBTU” for O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium     tetrafluoroborate, -   “TEA” for triethylamine, -   “TFA” for trifluoroacetic acid, and -   “THF” for tetrahydrofuran.

Abbreviations used in the Examples are defined as follows: “° C.” for degrees Celsius, “MS” for mass spectrometry, “ESI” for electrospray ionization mass spectroscopy, “HR” for high resolution, “LC-MS” for liquid chromatography mass spectrometry, “eq” for equivalent or equivalents, “g” for gram or grams, “h” for hour or hours, “mg” for milligram or milligrams, “mL” for milliliter or milliliters, “mmol” for millimolar, “M” for molar, “min” for minute or minutes, “rt” for room temperature, “NMR” for nuclear magnetic resonance spectroscopy, “tlc” for thin layer chromatography, “atm” for atmosphere, and “α”, “β”, “R”, “S”, “E”, and “Z” are stereochemical designations familiar to one skilled in the art.

“HPLC” is an abbreviation used herein for high pressure liquid chromatography. Reverse-phase HPLC can be carried out using a variety of columns with gradient elution from 0% to 100% buffer B in buffer A (buffer A: 90% water and 10% MeOH containing 0.1% trifluoroacetic acid, buffer B: 10% water, 90% MeOH containing 0.1% trifluoroacetic acid). If necessary, organic layers can be dried over sodium sulfate unless otherwise indicated. However, unless otherwise indicated, the following conditions are generally applicable. “LC-MS” refers to high pressure liquid chromatography carried out according to the definition for HPLC with a mass spectrometry detector.

Melting points were determined on a Mel-Temp II apparatus and are uncorrected. IR spectra were obtained on a single-beam Nicolet Nexus FT-IR spectrometer using 16 accumulations at a resolution of 4.00 cm−1 on samples prepared in a pressed disc of KBr or as a film on KBr plates. Proton NMR spectra (300 MHz, referenced to tetramethylsilane) were obtained on a Varian INOUA 300, Bruker Avance 300, Avance 400, or Avance 500 spectrometer. Data were referred to the lock solvent. Electrospray Ionization (ESI) experiments were performed on a Micromass II Platform single-quadrupole mass spectrometer, or on a Finnigan SSQ7000 mass spectrometer. HPLC analyses were obtained using the following columns with UV detection at 223 nm using a standard solvent gradient program as follows:

1 minute at 100% buffer A, followed by a gradient from 100% buffer A to 100% buffer B over 1-3 minutes, followed by 1 minute at 100% buffer B. The length of the gradient can be determined by the total run time less 2 minutes. Retention times (RT) and Run times (RunT) are reported as RT/RunT in the experimental section. The columns employed include:

Column 1: XTerra 4.6×30 mm S5

Column 2: Phenomenex-Luna 4.6×50 mm S10

Column 3: Phenomenex-Luna 4.6×30 mm S10

Prep HPLC solvent conditions: When described as performed under “standard conditions”, Samples were dissolved in acetonitrile (up to 100 mg/mL) and run using the following gradient program with a solvent flow rate of 40.0 mL/min on an Xterra prep 18 column.

Acetonitrile H₂O Time (min) (0.05% TFA) (0.05% TFA) Initial 10 90 10.0 70 30 10.01–15 90 10

Synthesis of Intermediates Intermediate A 3,5-Dichloro-4-aminobenzylamine

To lithium aluminum hydride (0.57 g, 15 mmol) in dry THF (20 mL) was added dropwise 3,5-dichloro-4-aminobenzonitrile (1.87 g, 10 mmol) in THF (30 mL). The mixture was stirred at rt for 2 h. Then, sodium sulfate decahydrate (4.83 g, 15 mmol) was added and the mixture was stirred for 30 min. The solid was filtered off and washed with THF three times. The solvent was removed under vacuum and the residue was purified by chromatography with Methanol/DCM (3:7) as the eluant. 3,5-Dichloro-4-aminobenzylamine was obtained as an off-white solid (1.5 g, 80%). ¹H NMR (300 MHz, CD₃OD): δ 3.65 (s, 2H), 7.2 (s, 2H).

Intermediate B 4-Acetamido-3,5-dichloro-benzylamine

Step B (1): To a solution of 3,5-dichloro-4-amino-benzonitrile (187 mg, 1 mmol) in 4 mL of THF at room temperature was added 2.2 mL of 1.0 M NaHMDS in THF. The resulting reaction mixture was stirred at room temperature for 30 min, at which time acetyl chloride (3.1 mmol) was added. DCM (100 mL) and water (100 mL) were added to the reaction mixture after being stirred overnight, followed by the addition of 5 mL of a 1.4 N HCl aqueous solution. The layers were separated and the aqueous layer was extracted with DCM (2×100 mL). The extracts were combined and solvents were evaporated in vacuo. The residue was purified by HPLC to give 4-acetamido-3,5-dichloro-benzonitrile. MS (ESI) (M−H)⁺=227.05. ¹H NMR (300 MHz, CDCl₃) δ 7.63 (s, 2H), 2.21 (s, 3H).

Step B (2): To a solution of 4-acetamido-3,5-dichloro-benzonitrile (114 mg, 0.5 mmol) in 5 mL of THF was added lithium aluminum hydride (120 mg, 3.2 mmol) in one portion and the resulting mixture was stirred for 3 h. Sodium sulfate decahydrate (1 g, 3.2 mmol) was added in one portion and the mixture was stirred for 30 min. The solid was filtered off and the filtrate was concentrated. The residue was purified by HPLC to give 4-acetamido-3,5-dichloro-benzylamine (35 mg, 30%). ¹H NMR (500 MHz, CD₃OD): δ 7.46 (s, 2H), 3.78 (s, 2H), 2.18 (s, 3H).

Intermediate C N—Boc-S-methylisothiourea

To a rapidly stirred suspension of S-Methylisothiourea hemisulfate (60.8 g, 0.437 mol) in CH₂Cl₂ (600 mL) was added 2N NaOH (300 mL, 0.6 mol). This mixture was cooled to 0° C. on an ice bath, and a solution of di-tert-butyl dicarbonate (43.2 g, 0.198 mol) was added dropwise over 6 h. Upon completion of the addition, the mixture was stirred an additional 20 min, diluted with IL of CH₂Cl₂ and the phases were separated. The organic portion was washed with water (2×500 ml) and dried over Na₂SO₄. Filtration and concentration provided the desired N—Boc-S-methylisothiourea as a white solid (35.5 g, 0.187 mol, 94% yield based on Boc₂O).

Intermediate D (S)-tert-Butyl methylthio-N-(1-phenylpyrrolidine-5-carbonyl)carbonoimidoylcarbamate

Step D (1): To a sealed tube flushed with nitrogen was added L-proline (3.684 g, 32.0 mmol), potassium carbonate (6.634 g, 48.0 mmol), copper (I) iodide (609 mg, 3.2 mmol), iodobenzene (3.58 mL, 32.0 mmol) and N,N-dimethylacetamide. The mixture was heated at 90° C. for 48 hours, then cooled to room temperature. Water was added, and the pH adjusted to <3 with concentrated HCl. The aqueous phase was extracted 4 times with ethyl acetate. The combined organic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated in vacuo. Silica gel chromatography (0 to 100% EtOAc/hexane gradient) afforded N-phenylproline which was used directly in the next step. LC-MS (M+H)⁺=192. LC-MS RT (column 1)=2.230/4.00. ¹H NMR (500 MHz, CDCl₃): δ 10.574 (br s, 1H), 7.29-7.25 (m, 2H), 6.785 (t, J=7.5 Hz, 1H), 6.613 (d, J=8.0 Hz, 2H), 4.25 (dd, J=2.4, 8.8 Hz, 1H), 3.60 (dt, J=2.8, 8.3 Hz, 1H), 3.36 (m, 1H), 2.38-2.01 (m, 4H).

Step D (2): To a solution of the product from step D(1) (151 mg, 791 umol) and Intermediate C (158 mg, 834 umol) in DMF (7.3 mL) was added HATU (381 mg, 1.0 mmol), then NMM (321 uL, 2.92 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with ethyl acetate and washed with 1 M HCl, water, and brine. The organic layer was then dried with magnesium sulfate, filtered and concentrated in vacuo. Silica gel chromatography (0 to 50% EtOAc/hexane gradient) afforded (S)-tert-butyl methylthio-N-(1-phenylpyrrolidine-5-carbonyl)carbonoimidoylcarbamate (231.2 mg, 81%). LC-MS (M+H)⁺=364. LC-MS RT (column 1)=3.020/4.00. ¹H NMR (500 MHz, CD₃OD): δ 7.21 (t, J=7.9 Hz, 2H), 6.75 (t, J=7.3 Hz, 1H), 6.60 (d, J=7.9 Hz, 2H), 4.08 (dd, J=2.1, 9.5 Hz, 1H), 3.81 (t, J=6.9 Hz, 1H), 3.32-3.25 (m, 1H), 2.44-2.33 (m, 1H), 2.32 (s, 3H), 2.22-1.98 (m, 3H), 1.34 (s, 9H). ¹³C NMR (500 MHz, CD₃OD): 174.41, 168.05, 160.33, 147.15, 129.30, 118.25, 112.99, 81.15, 78.47, 64.43, 49.34, 31.43, 27.34, 27.15, 24.10, 13.36.

Intermediate E (S)-tert-Butyl N-(1-(4-methoxyphenyl)pyrrolidine-5-carbonyl)(methylthio)carbonoimidoylcarbamate

Step E (1): In a 3-necked flask, L-proline (17.25 g, 150 mmol), p-bromoanisole (19.4 mL, 155 mmol), potassium carbonate (21.4 g, 155 mmol), and copper(II) oxide (1.00 g, 12.6 mmol) were combined. DMF (25 mL) was added, the vessel flushed with argon, and the reaction heated in a 170° C. oil bath overnight. The reaction was cooled to room temperature, water was added, and the mixture filtered through Celite. The aqueous filtrate was extracted three times with methylene chloride (discarded). The aqueous phase was then adjusted to pH 2.5 with concentrated HCl. The desired material fell to the bottom of the separtory funnel as a dense brown oil. The brown oil turned to solid under a stream of nitrogen, and was used without further purification in the subsequent reaction. LC-MS (M+H)⁺=222. LC-MS RT (column 3)=1.563/5.00.

Step E (2): The product from step E(1) above was subjected to the reaction conditions of step D(2) to afford Intermediate E in 60% yield. LC-MS (M+H)⁺=394. LC-MS RT (column 2)=4.010/5.00. ¹H NMR (500 MHz, CDCl₃): δ 6.79 (d, J=9.2 Hz, 2H), 6.52 (d, J=8.5 Hz, 2H), 3.99 (d, J=7.9 Hz, 1H), 3.81-3.75 (m, 1H), 3.70 (s, 3H), 3.21 (q, J=8.3 Hz, 1H), 2.37-2.26 (m, 4H), 2.23-2.15 (m, 1H), 2.08-1.98 (m, 2H), 1.33 (s, 9H).

Intermediate F N-(4-(Aminomethyl)-2,6-dichlorophenyl)-2-(dimethylamino)acetamide

Step F (1): To a solution of the compound of Intermediate A (9 g, 0.047 mol) in dry THF under N₂, diisopropyl ethylamine (8.2 ml, 0.047 mol) was added. The reaction mixture was cooled to 0° C. and then Boc anhydride (10.26 g, 0.047 mol) dissolved in dry THF (25 mL) was added drop wise maintaining the reaction temperature at 0° C. After the addition was over, the reaction mixture was allowed to reach room temperature and stirred for about 2 hrs. After ensuring the absence of the starting material, THF was removed under the vacuum and the resulting solid was dissolved in ethyl acetate. The organic layer was washed with water (25 mL), brine (25 mL), dried over anhydrous sodium sulfate and concentrated under vacuum to afford the product as pale yellow colored solid. The product was further purified by recrystallization (9.9 g, 72.2%) using pet ether/ether.

Step F (2): To a suspension of NaH (1.37 g, 0.034 mol) in dry DMF (5 mL) under N₂, the compound of step F (1) (10 g, 0.034 mol) was added at 0° C. The reaction mixture was stirred for about 30 min. at room temperature. The reaction mixture was again cooled to 0° C. and bromoacetyl bromide (7.62 g, 0.037 mol) was added and allowed to stir overnight at room temperature. The reaction mixture was poured on to crushed ice and the precipitate was extracted with ethyl acetate (3×50 ml). The organic layer was washed with water (25 ml), brine (25 ml), dried over anhydrous sodium sulfate and concentrated under vacuum. The resulting product was purified by column chromatography using CHCl₃:MeOH (9:1) as eluent to afford the product as brown colored solid (6 g, 42.5%)

Step F (3): To a solution of the compound of step F (2) (6 g 0.014 mol) in dry DMF (60 ml) under N₂, anhydrous K₂CO₃ (6.03 g, 0.043 mol) was added with stirring. The reaction mixture was cooled to 0° C. and N,N-dimethylamine hydrochloride (2.37 g, 0.029 mol) was added at once. The reaction mixture was allowed stir for overnight at room temperature. The reaction mixture was poured into water and extracted with ethyl acetate (2×150 ml). The organic layer was washed with water (25 ml), brine (25 ml), dried over anhydrous sodium sulfate and concentrated under vacuum to remove the volatiles. The resulting crude product was purified by column chromatography using CHCl₃: MeOH (9:1) as eluent to afford the product as an off-white solid (4 g, 72.9%)

Step F (4): To a solution of the compound of step F (3) (12 g) in dry dioxane (50 ml), HCl in dioxane (100 ml) was added while stirring. The reaction mixture was heated to 50° C. overnight. After ensuring the absence of the starting material, the reaction mixture was concentrated under vacuum to remove the dioxane and the obtained solid was washed with pet ether/ether mixture to provide the pure product as a white solid in its HCl salt form (9 g, 90.4%) ¹H NMR (400 MHz, (CD₃)₂SO): δ 11.05 (s, 1H), 10.2 (br s, 1H), 8.63 (br s, 2H), 7.75 (s, 2H), 4.24 (s, 2H), 4.05 (s, 2H), 2.87 (s, 6H).

Intermediate G N-(4-(Aminomethyl)-2-chloro-6-methylphenyl)-2-(dimethylamino)acetamide

The title compound was prepared as outlined in the scheme following the general procedures for the preparation of Intermediate F.

LC/MS Method A: Column: XTERRA 4.6×30 mm S5; Flow Rate: 4 mL/min.; Solvent A: 10% MeOH—90% water—0.1% TFA; Solvent B: 90% MeOH—10% water—0.1% TFA; Gradient: % B 0-100; Gradient Time: 3 min.

Step G (1): 4-(N—Boc-Aminomethyl)-2-chloro-6-methylbenzenamine

¹H NMR (500 MHz, CDCl₃) δ 7.06 (s, 1H), 6.88 (s, 1H), 4.15 (s, 2H), 2.18 (s, 3H), 1.45 (s, 9H); LC/MS RT (Method A)=2.841 min.; (M+H)⁺=271 (base peak 215).

Step G (2): N-(4-(N—Boc-Aminomethyl)-2-chloro-6-methylphenyl)-2-bromoacetamide

¹H NMR (500 MHz, CDCl₃) δ 7.87 (s, 1H), 7.21 (s, 1H), 7.07 (s, 1H), 4.85 (s, 1H), 4.25 (d, J=5.5 Hz, 2H), 4.06 (s, 2H), 2.25 (s, 3H), 1.46 (s, 9H); LC/MS RT (Method A)=2.866 min.; (M+H)⁺=none (base peak 369.04).

Step G (3): N-(4-(N—Boc-Aminomethyl)-2-chloro-6-methylphenyl)-2-(dimethylamino)acetamide

¹H NMR (500 MHz, CDCl₃) δ 8.82 (s, 1H), 7.18 (s, 1H), 7.04 (s, 1H), 4.91 (s, 1H), 4.22 (d, J=5.5 Hz, 2H), 3.12 (s, 2H), 2.43 (s, 6H), 2.24 (s, 3H), 1.44 (s, 9H); LC/MS RT (Method A)=2.330 min.; (M+H)⁺=356.2.

Step G (4): N-(4-(Aminomethyl)-2-chloro-6-methylphenyl)-2-(dimethylamino)-acetamide

The title compound was prepared from 4-(aminomethyl)-2-chloro-6-methylbenzenamine following the general procedures as described for the preparation of Intermediate F. ¹H NMR (500 MHz, CD₃OD) δ 7.48 (s, 1H), 7.35 (s, 1H), 4.28 (s, 2H), 4.10 (s, 2H), 3.01 (s, 6H), 2.31 (s, 3H); LC/MS (Method A) RT=0.193 min.; (M+H)⁺=256.

Intermediate H (S)-tert-Butyl N-(1-pyrrolidine-2-carbonyl-4-trans-methoxy)(methylthio)carbonoimidoylcarbamate

Step H (1): To a suspension of trans-L-4-hydroxy proline (28.17 g, 215 mmol) in 2:1 THF:H₂O (700 mL) was added 2.5 M NaOH (118 mL), then Boc₂O (67.1 mL, 292 mmol). The reaction was stirred overnight at room temperature. The THF was then removed in vacuo, and 10% KHSO₄ solution (423 ml) was added. The mixture was extracted three times with ethyl acetate. The combined organic layers were partitioned with brine, dried over MgSO₄, and concentrated to afford trans-L-N—Boc-4-hydroxy proline (44.41 g, 89% yield). ¹H NMR (500 MHz, DMSO-d₆) δ ppm 12.49 (s, 1H), 4.86-5.23 (m, 1H), 4.20-4.30 (m, 1H), 4.07-4.16 (m, 1H), 3.31-3.46 (m, 1H), 3.19-3.29 (m, 1H), 2.03-2.18 (m, 1H), 1.81-1.95 (m, 1H), 1.39 (s, 3H), 1.34 (s, 6H).

Step H (2): The material from step H (1) (9.02 g, 39.1 mmol) in DMF (66.7 mL) was slowly added to a 0° C. suspension of NaH (3.90 g, 97.5 mmol as a 60% dispersion in oil) in DMF (83.5 mL). After the addition was complete (˜45 minutes), the reaction was stirred an additional 15 min at 0° C., then for 30 min at room temperature. The reaction was again cooled to 0° C., and iodomethane (2.43 ml, 39.0 mmol) was added. The reaction was allowed to come to room temperature, and stirred at this temperature for 2 h. The reaction was carefully quenched with 1 M HCl (100 mL). The mixture was extracted three times with EtOAc. The combined organics were washed with water, then brine, and dried over MgSO₄. The extracts were concentrated in vacuo then subjected to silica gel chromatography (0 to 20% MeOH/CHCl₃ gradient) to afford the desired trans-L-N—Boc-4-methoxy proline (6.19 g, 65% yield).

Step H (3): To a solution of the product from step H (2) (6.19 g, 25.5 mmol) in dichloromethane (112 mL) was added TFA (12.6 mL). After 3 h, the volatiles were removed in vacuo. The residue was taken up in pH 7 buffer, then lyophilized to produce trans-L-4-methoxy proline, which was used directly in the subsequent experiment.

Step H (4): trans-L-4-methoxy proline (step H (3)) was reacted in the manner described in step E (1) but using bromobenzene to produce trans-L-N-phenyl-4-methoxy proline (2.667 g, 48% yield). LC-MS (M+H)⁺=222. LC-MS RT (column 1)=1.790/5.00.

Step H (5): trans-L-N-phenyl-4-methoxy proline (step H (4)) (2.667 g, 12.07 mmol) was reacted as described in step D (2) to produce the desired (S)-tert-butyl N-(1-pyrrolidine-2-carbonyl-4-trans-methoxy)(methylthio)-carbonoimidoylcarbamate (3.99 g, 84% yield). LC-MS (M+H)⁺=294. LC-MS RT=3.301/5.00. ¹H NMR (500 MHz, CDCl₃) δ ppm 12.56 (s, 0.7H), 12.19 (s, 0.3H), 7.12-7.23 (m, 2H), 6.65-6.83 (m, 1H), 6.48-6.61 (m, 2H), 4.12-4.55 (m, 2H), 3.97-4.06 (br s, 0.7H), 3.80 (br s, 0.3H), 3.37-3.48 (m, 1H), 3.31-3.36 (s, 3H), 2.25-2.55 (m, 5H), 1.28-1.55 (m, 9H).

Intermediate I cis-tert-Butyl N-(3-phenyl-3-azabicyclo[3.1.0]hexane-2-carbonyl)(methylthio)carbonoimidoylcarbamate

Step I (1): In a sealed tube, cis-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (547 mg, 4.3 mmol), iodobenzene (496 μL, 4.43 mmol), potassium carbonate (611 mg, 4.43 mmol), and copper(II) oxide (41 mg, 0.516 mmol) were combined. DMF (1.1 mL) was added, the vessel flushed with nitrogen, and the reaction heated in a 170° C. oil bath overnight. The reaction was cooled to room temperature, water was added, and the mixture filtered through Celite. The aqueous filtrate was extracted three times with methylene chloride (discarded). The aqueous phase was then adjusted to pH 2.5 with concentrated HCl and extracted three times with ethyl acetate. The combined ethyl acetate extracts were washed with saturated aqueous sodium bicarbonate, dried (MgSO₄), filtered, and concentrated in vacuo to give a brown oil (263 mg, 30%) which was used without further purification in subsequent reaction. LC-MS (M+H)⁺=204. LC-MS RT (column 2)=1.688/3.000.

Step I (2): The product from step I (1) above was subjected to the reaction conditions of step D (2) to afford Intermediate I in 42% yield. LC-MS (M+H)⁺=376. LC-MS RT (column 2)=2.247/3.000. ¹H NMR (300 MHz, CDCl₃) δ ppm 7.13-7.21 (m, 2H), 6.79 (t, J=7.50 Hz, 1H), 6.59 (d, J=8.05 Hz, 2H), 4.22 (br s, 1H), 3.96 (d, J=8.78 Hz, 1H), 3.38 (dd, J=8.78, 4.76 Hz, 1H), 2.36 (s, 3H), 2.04-2.12 (m, 1H), 1.80 (ddd, J=11.62, 7.78, 4.39 Hz, 1H), 1.35 (s, 9H), 1.25 (t, J=7.14 Hz, 1H), 0.85 (br s, 1H), 0.69-0.78 (m, 1H)

EXAMPLE 1 (S)—N-((4-Amino-3,5-dichlorobenzylamino)(amino)methylene)-1-phenylpyrrolidine-2-carboxamide

Step 1 (A): (S)-tert-Butyl N-(4-amino-3,5-dichlorobenzyl)-N′-(1-phenylpyrrolidine-5-carbonyl)carbamimidoylcarbamate

To a suspension of Intermediate D (150 mg, 413 umol) and Intermediate A (157.8 mg, 826 umol) in CH₂Cl₂ (6.4 mL) was added DIPEA (143 uL, 821 umol). The mixture was stirred at rt overnight. Solvents were removed in vacuo. The residue was loaded onto a silica gel chromatography column, and eluted with a 0 to 75% EtOAc/hexane gradient to afford the pure product (181.8 mg, 87% yield). LC-MS (M+H)⁺=506. LC-MS RT (column 1)=2.806/4.00. ¹H NMR (500 MHz, CD₃OD, rotamers seen): 67.23-7.15 (m, 3.3H), 7.14-7.08 (m, 0.7H), 6.73 (t, J=7.3 Hz, 0.7H), 6.63-6.55 (m, 1.7H), 6.49 (d, J=7.9 Hz, 0.6H), 4.42-4.30 (m, 2H), 4.19-4.14 (dd, J=2.4, 8.9 Hz, 0.3H), 4.11-4.06 (dd, J=2.4, 9.5 Hz, 0.7 Hz), 3.86-3.80 (m, 0.7H), 3.56-3.48 (m, 0.3H), 3.31-3.24 (m, 1H), 2.41-2.28 (m, 1H), 2.22-2.14 (m, 1H), 2.11-2.03 (m, 2H), 1.47 (s, 2.7H), 1.34 (s, 6.3H). ¹³C NMR (500 MHz, CD₃OD, rotamers seen): δ 177.56, 163.08, 155.35, 147.07, 140.60, 129.23, 128.94, 127.74, 127.60, 127.53, 119.27, 118.03, 115.86, 112.90, 112.11, 83.69, 79.56, 78.45, 65.96, 64.55, 49.16, 43.28, 43.15, 31.56, 31.47, 27.44, 27.21, 24.01, 23.91.

Step 1 (B): (S)—N-((4-Amino-3,5-dichlorobenzylamino)(amino)-methylene)-1-phenylpyrrolidine-2-carboxamide

To a solution of the product of step (A) (81.3 mg, 161 umol) in CH₂Cl₂ (3.6 mL) was added TFA (360 uL). The mixture was stirred at rt for 2.5 h, and then the mixture was concentrated to give the title compound after prep HPLC. LC-MS (M+H)⁺=406. LC-MS RT (column 1)=2.035/4.00. ¹H NMR (500 MHz, CD₃OD): δ 7.34-7.18 (m, 4H), 6.83 (t, J=7.3 Hz, 1H), 6.67 (d, J=7.9 Hz, 2H), 4.34 (s, 2H), 4.17 (dd, J=2.8, 9.8 Hz, 1H), 3.76 (t, J=7.0 Hz, 1H), 3.28-3.21 (m, 1H), 2.52-2.37 (m, 1H), 2.32-2.18 (m, 1H), 2.17-1.98 (m, 2H). ¹³C NMR (500 MHz, CD₃OD): δ 177.99, 153.59, 147.59, 141.62, 129.42, 127.68, 123.76, 119.34, 118.84, 113.35, 93.76, 64.88, 49.84, 48.83, 44.10, 31.55, 24.02.

EXAMPLE 2 (S)—N-((4-Acetamido-3,5-dichlorobenzylamino)(amino)methylene)-1-phenylpyrrolidine-2-carboxamide

Step 2 (A): (S)-tert-Butyl N-(4-acetamido-3,5-dichlorobenzyl)-N′-(1-phenylpyrrolidine-5-carbonyl)carbamimidoylcarbamate

To a suspension of Intermediate D (78.2 mg, 215 umol) and Intermediate B (100.3 mg, 431 umol) in CH₂Cl₂ (3.4 mL) was added DIPEA (74.2 uL, 426 umol). The mixture was stirred at rt overnight. Solvents were removed in vacuo. The residue was loaded onto a silica gel chromatography column, and eluted with a 0% to 75% EtOAc/hexane gradient to afford the pure product (94 mg, 76% yield). LC-MS (M+H)⁺=548. LC-MS RT (column 2)=2.062/3.00.

Step 2 (B): (S)—N-((4-Acetamido-3,5-dichlorobenzylamino)(amino)-methylene)-1-phenylpyrrolidine-2-carboxamide

To a solution of the product of step (A) (89 mg, 162 umol) in CH₂Cl₂ (3.6 mL) was added TFA (360 uL). The mixture was stirred at rt for 2.5 h, and then the mixture was concentrated to give the title compound after prep HPLC. LC-MS (M+H)⁺=448. LC-MS RT (column 1)=2.313/4.00. ¹H NMR (500 MHz, CD₃OD): δ 7.47 (s, 2H), 7.26 (d, J=7.9 Hz, 2H), 6.82 (t, J=7.3 Hz, 1H), 6.68 (d, J=8.2 Hz, 2H), 4.53 (s, 2H), 4.20 (dd, J=2.4, 9.8 Hz, 1H), 3.78 (t, J=6.4 Hz, 1H), 3.31-3.25 (m, 1H), 2.52-2.39 (m, 1H), 2.31-2.24 (m, 1H), 2.19 (s, 3H), 2.15-2.04 (m, 2H). ¹³H NMR (500 MHz, CD₃OD): δ 217.97, 178.02, 171.26, 154.20, 147.55, 136.94, 134.95, 129.42, 127.48, 118.70, 113.30, 64.72, 49.79, 43.80, 31.54, 24.01, 21.33.

EXAMPLE 3 (S)—N-((3,5-Dichloro-4-(2-(dimethylamino)-acetamido)-benzylamino)(amino)methylene)-1-phenylpyrrolidine-2-carboxamide

Step 3 (A): (S)-tert-Butyl N-(3,5-dichloro-4-(2-(dimethylamino)-acetamido)benzyl)-N′-(1-phenylpyrrolidine-5-carbonyl)-carbamimidoylcarbamate

To a solution of Example 1, step (A) (55.6 mg, 110 umol) in CH₂Cl₂ (1.5 mL) under nitrogen was added triethylamine (17.5 uL, 126 umol), followed by bromoacetyl bromide (10.6 uL, 121 umol). After 1 h, the reaction was ˜30% complete by TLC. Another aliquot of bromoacetyl bromide 10.6 uL, 121 umol) was added. After another 1 h, TLC revealed no starting material. Solvents were removed in vacuo. The crude material was used without further purification in the next step. LC-MS (M+H)⁺=626. LC-MS RT (column 1)=2.705/4.00.

Step 3 (B): To a solution of the product of step (A) (110 umol) in CH₂Cl₂ (1.5 mL) was added 2M dimethylamine in THF (1.6 mL, 3.2 mmol). After 2 h, a precipitate had formed, and the reaction was complete by LC/MS. Solvents were removed in vacuo. The residue was loaded onto a silica gel chromatography column, and eluted with a 0 to 100% EtOAc/hexane gradient to afford the pure product (53.3 mg, 82% yield). LC-MS (M+H)⁺=591. LC-MS RT (column 1)=2.372/4.00.

Step 3 (C): (S)—N-((3,5-dichloro-4-(2-(dimethylamino)acetamido)-benzylamino)(amino)methylene)-1-phenylpyrrolidine-2-carboxamide

To a solution of the product of step (B) (82.6 mg, 140 umol) in CH₂Cl₂ (3.1 mL) was added TFA (310 uL). The mixture was stirred at rt for 2.5 h, and then the mixture was concentrated to give the title compound after prep HPLC. LC-MS (M+H)⁺=491. LC-MS RT (column 1)=1.445/4.00. ¹H NMR (500 MHz, CD₃OD): δ 7.53 (s, 2H), 7.26 (t, J=7.9 Hz, 2H), 6.81 (t, J=7.3 Hz, 1H), 6.67 (d, J=7.9 Hz, 2H), 4.57 (s, 2H), 4.30 (s, 2H), 4.21 (dd, J=2.7, 9.8 Hz, 1H), 3.82-3.75 (m, 1H), 3.32-3.25 (m, 1H), 3.06-2.99 (s, 6H), 2.51-2.40 (m, 1H), 2.31-2.24 (m, 1H), 2.15-2.04 (m, 2H). ¹³C NMR (500 MHz, CD₃OD): δ 178.06, 163.85, 154.29, 147.54, 137.75, 134.65, 131.37, 129.39, 127.64, 118.64, 113.28, 64.68, 58.09, 49.77, 43.77, 43.45, 31.52, 23.98.

EXAMPLE 4 (S)—N-((4-Amino-3,5-dichlorobenzylamino)(amino)methylene)-1-(4-methoxyphenyl)pyrrolidine-2-carboxamide

Step 4 (A): (S)-tert-Butyl N-(4-amino-3,5-dichlorobenzyl)-N′-(1-(4-methoxyphenyl)pyrrolidine-5-carbonyl)carbamimidoylcarbamate

To a suspension of Intermediate E (417.5 mg, 1.06 mmol) and Intermediate A (404.7 mg, 2.12 mmol) in CH₂Cl₂ (16.5 mL) was added DIPEA (366 uL, 2.10 mmol). The mixture was stirred at rt overnight. Solvents were removed in vacuo. The residue was loaded onto a silica gel chromatography column, and eluted with a 0 to 75% EtOAc/hexane gradient to afford the pure product (510.8 mg, 90% yield). LC-MS (M+H)⁺=536. LC-MS RT (column 2)=2.083/3.00. ¹H NMR (500 MHz, CDCl₃, major rotamer): δ 7.14 (s, 2H), 6.82 (d, J=9.2 Hz, 2H), 6.53 (d, J=8.9 Hz, 2H), 4.45-4.42 (m, 2H), 4.01 (dd, J=2.3, 9.6 Hz, 1H), 3.86-3.80 (m, 1H), 3.74-3.71 (s, 3H), 3.28-3.20 (m, 1H), 2.39-2.26 (m, 1H), 2.22-2.04 (m, 3H), 1.37 (s, 9H).

Step 4 (B): (S)—N-((4-Amino-3,5-dichlorobenzylamino)-(amino)methylene)-1-(4-methoxyphenyl)pyrrolidine-2-carboxamide

To a solution of the product of step (A) (160.6 mg, 299.7 umol) in CH₂Cl₂ (6.6 mL) was added TFA (660 uL). The mixture was stirred at rt for 2.5 h, and then the mixture was concentrated to give the title compound after prep HPLC. LC-MS (M+H)⁺=436. LC-MS RT (column 1)=2.425/5.00. ¹H NMR (500 MHz, CD₃OD): δ 7.24 (s, 2H), 6.87 (d, J=8.9 Hz, 2H), 6.62 (d, J=8.9 Hz, 2H), 4.34 (s, 2H), 4.06 (dd, J=2.8, 9.8 Hz, 1H), 3.78-3.71 (m, 4H), 3.24-3.14 (m, 1H), 2.48-2.36 (m, 1H), 2.27-2.19 (m, 1H), 2.13-1.98 (m, 2H). ¹³C NMR (500 MHz, CD₃OD): δ 178.35, 153.46, 142.01, 141.56, 127.69, 123.87, 119.34, 115.05, 114.37, 65.31, 55.15, 50.45, 44.05, 31.62, 24.15.

EXAMPLE 5 (S)—N-((4-Acetamido-3,5-dichlorobenzylamino)(amino)methylene)-1-(4-methoxyphenyl)pyrrolidine-2-carboxamide

Step 5 (A): (S)-tert-Butyl N-(4-acetamido-3,5-dichlorobenzyl)-N′-(1-(4-methoxyphenyl)pyrrolidine-5-carbonyl)carbamimidoylcarbamate

To a suspension of Intermediate E (146.8 mg, 373.5 umol) and Intermediate B (174.0 mg, 747 umol) in CH₂Cl₂ (5.8 mL) was added DIPEA (128.7 uL, 739 umol). The mixture was stirred at rt overnight. Solvents were removed in vacuo. The residue was loaded onto a silica gel chromatography column, and eluted with a 0 to 75% EtOAc/hexane gradient to afford the pure product (168.1 mg, 78% yield) plus some recovered starting material. LC-MS (M+H)⁺=578. LC-MS RT (column 1)=2.425/4.00. ¹H NMR (500 MHz, CD₃OD, rotamers seen): δ 7.33-7.21 (m, 2H), 6.81 (d, J=8.9 Hz, 1.4H), 6.74 (d, J=9.2 Hz, 0.6H), 6.53 (d, J=9.2 Hz, 1.4H), 6.47-6.36 (m, 0.6H), 4.62-4.38 (m, 2H), 4.10 (q, J=7.0 Hz, 1.3H), 4.02 (dd, J=2.1, 9.5 Hz, 0.7H), 3.86-3.79 (m, 0.8H), 3.73-3.70 (m, 3.2H), 3.32-3.19 (m, 1H), 2.39-2.28 (m, 0.7H), 2.27-2.03 (m, 5.3H), 1.44 (s, 2.8H), 1.35 (s, 6.2H).

Step 5 (B): (S)—N-((4-acetamido-3,5-dichlorobenzylamino)(amino)-methylene)-1-(4-methoxyphenyl)pyrrolidine-2-carboxamide

To a solution of the product of step (A) (168 mg, 291 umol) in CH₂Cl₂ (6.4 mL) was added TFA (640 uL). The mixture was stirred at rt for 2.5 h, and then the mixture was concentrated in vacuo. Prep HPLC, followed by flash chromatography (silica gel, MeOH/chloroform gradient) afforded the title compound. LC-MS (M+H)⁺=478. LC-MS RT (column 2)=2.115/5.00. ¹H NMR (500 MHz, CD₃OD): δ 7.39 (s, 2H), 6.77 (d, J=6.7 Hz, 2H), 6.49 (br s, 2H), 4.42 (s, 2H), 4.00 (d, J=6.4 Hz, 1H), 3.71 (s, 3H), 3.62-3.44 (m, 1H), 3.25 (q, J=7.7 Hz, 1H), 2.37-2.23 (m, 1H), 2.19 (s, 3H), 2.14-1.90 (m, 3H).

EXAMPLE 6 (S)—N-((3,5-Dichloro-4-(2-(dimethylamino)acetamido)benzylamino)-(amino)methylene)-1-(4-methoxyphenyl)pyrrolidine-2-carboxamide

Step 6 (A): To a solution of Example 1, step (A) (200.0 mg, 373.2 umol) in CH₂Cl₂ (4.9 mL) under nitrogen was added triethylamine (59.5 uL, 429 umol), followed by bromoacetyl bromide (35.9 uL, 411 umol). After 1 h, the reaction was ˜30% complete by TLC. Another aliquot of bromoacetyl bromide 35.9 uL, 411 umol) was added. After an additional hour, the reaction was ˜80% complete by TLC. Another aliquot of bromoacetyl bromide 18 uL, 205 umol) was added After another 1 h, TLC revealed no starting material. Solvents were removed in vacuo. The crude material was used without further purification in the next step.

Step 6 (B): (S)-tert-Butyl N-(3,5-dichloro-4-(2-(dimethylamino)-acetamido)benzyl)-N′-(1-(4-methoxyphenyl)pyrrolidine-5-carbonyl)carbamimidoylcarbamate

To a solution of the product from step (A) (373 umol) in CH₂Cl₂ (4.9 mL) was added 2M dimethylamine in THF (4.9 mL, 9.8 mmol). After stirring overnight, a precipitate had formed, and the reaction was complete by LC/MS. Solvents were removed in vacuo. The residue was loaded onto a silica gel chromatography column, and eluted with a 0 to 100% EtOAc/hexane gradient to afford the pure product (131.9 mg, 57% yield). LC-MS (M+H)⁺=621. LC-MS RT (column 1)=2.758/5.00. ¹H NMR (500 MHz, CD₃OD, rotamers seen): δ 7.45 (s, 1.5H), 7.39 (s, 0.5H), 6.83 (d, J=8.9 Hz, 1.5H), 6.74 (d, J=8.9 Hz, 0.5H), 6.74 (d, J=8.9 Hz, 1.5H), 6.41 (d, J=8.9 Hz, 0.5H), 4.61-4.45 (m, 2H), 4.26-4.20 (dd, J=3.5, 5.7 Hz, 0.3H), 4.15-4.00 (m, 1.7H), 3.87-3.79 (m, 0.7H), 3.74-3.68 (m, 3H), 3.49-3.41 (m, 0.3H), 3.29-3.22 (m, 1H), 3.21-3.18 (s, 2H), 2.49-2.41 (m, 6H), 2.41-2.34 (m, 0.6H), 2.24-2.16 (m, 0.7H), 2.12-2.04 (m, 1.7H), 1.51 (s, 2H), 1.34 (s, 7H).

Step 6 (C): (S)—N-((3,5-dichloro-4-(2-(dimethylamino)acetamido)-benzylamino)(amino)methylene)-1-(4-methoxyphenyl)pyrrolidine-2-carboxamide

To a solution of the product of step (B) (131.9 mg, 212.4 umol) in CH₂Cl₂ (4.7 mL) was added TFA (470 uL). The mixture was stirred at rt for 3 h, and then the mixture was concentrated in vacuo. The residue was dissolved in CH₂Cl₂, and partitioned with saturated NaHCO₃. The organic phase was dried over magnesium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (0 to 25% MeOH/chloroform gradient) afforded the title compound. LC-MS (M+H)⁺=521. LC-MS RT (column 2)=1.622/5.00. ¹H NMR (500 MHz, CD₃OD): δ 7.35 (s, 2H), 6.75 (br s, 2H), 6.45 (br s, 2H), 4.39 (s, 2H), 4.06-3.90 (m, 1H), 3.70 (s, 3H), 3.62-3.41 (m, 1H), 3.28-3.14 (m, 3H), 2.45 (s, 6H), 2.36-2.20 (br s, 1H), 2.16-1.86 (m, 3H). ¹³C NMR (500 MHz, CD₃OD): δ 171.15, 142.93, 127.16, 114.84, 112.90, 78.45, 66.33, 62.56, 55.40, 45.20, 43.13, 31.90, 24.09.

EXAMPLES 7-30

The compounds of Examples 7-30 were prepared in a manner similar to that described for the synthesis of Example 3 (Scheme 3) but using the appropriate amine. The examples were purified by HPLC under standard conditions to provide the final products.

Ex- MS HPLC Ret. ample R₁₀R₁₁N— MW Observed Time (min) 7 cyclopropylamino 483.0 483.2 3.60 8 cyclopentylamino 511.1 511.2 3.89 9 4-methylcyclohexylamino 539.1 539.2 4.38 10 N-methylcyclohexylamino 539.1 539.2 4.15 11 azetidino 483.0 483.1 3.62 12 pyrrolidino 497.0 497.1 3.64 13 homopiperazino 526.1 526.2 3.39 14 morpholino 513.0 513.2 3.80 15 thiomorpholino 529.1 529.1 4.15 16 decahydroquinolino 565.2 565.2 4.40 17 isopropylamino 485.0 485.2 3.62 18 methylamino 457.0 457.1 3.47 19 benzylamino 533.1 533.2 4.16 20 4-methylbenzylamino 547.1 547.2 4.42 21 ethylamino 471.0 471.2 3.53 22 2-(N,N- 514.1 514.2 2.96 dimethylamino)ethylamino 23 2-methoxyethylamino 501.0 501.1 3.59 24 2-hydroxyethylamino 487.0 487.1 3.45 25 phenethylamino 547.1 547.2 4.37 26 N-methybenzylamino 547.1 547.2 4.35 27 N-methylbutylamino 513.1 513.2 4.01 28 N-methylisopropylamino 499.1 499.2 3.64 29 2-(thiophen-2-yl)ethylamino 553.1 553.2 4.26 30 cyclohexylmethylamino 539.1 539.2 4.48

EXAMPLE 31 (R)—N-(Amino(3,5-dichloro-4-(2-(dimethylamino)acetamido)-benzylamino)methylene)-1-(4-(trifluoromethyl)phenyl)pyrrolidine-2-carboxamide

Step 31 (A): (R)-tert-Butyl 1-(4-(trifluoromethyl)phenyl)pyrrolidine-2-carboxylate

A suspension of 4-(trifluoromethyl)phenylboronic acid (190 mg), copper (II) acetate (27 mg) and 4 Å molecular sieves (188 mg) in dichloromethane (4 mL) was stirred at room temperature for 5 min, and (R)-tert-butyl pyrrolidine-2-carboxylate (86 mg) in acetonitrile (0.5 mL) was added followed by triethylamine (0.07 mL). The reaction vial was flushed with oxygen, sealed and stirred at 50° C. for 24 h. The reaction mixture was filtered through a pad of Celite, and the filtrate was evaporated in vacuo. The residue was purified by preparative TLC eluting with 10% ethyl acetate/90% hexanes to give the title compound as a colorless oil (43 mg). HPLC retention time: 1.99 min (method A). MS (ESI) (M+H)⁺ 316.14. ¹H NMR (400 MHz, CDCl₃) δ 7.41 (1H, d, J=8.4 Hz), 6.52 (1H, d, J=8.4 Hz), 4.15 (1H, m), 3.53 (1H, m), 3.40 (1H, m), 2.0-2.3 (3H, m), 1.41 (9H, s).

Step 31 (B): (R)-1-(4-(Trifluoromethyl)phenyl)pyrrolidine-2-carboxylic acid

To a solution of (R)-tert-butyl 1-(4-(trifluoromethyl)phenyl)pyrrolidine-2-carboxylate (40 mg) in dichloromethane (0.10 mL) was added TFA (0.10 mL), and the resulting solution was stirred at room temperature for 5.5 h. The solvents were removed to give the title compound as the TFA salt. This crude product was used in step (C) without purification. HPLC retention time: 1.88 min (method A). MS (ESI) (M+H)⁺ 260.09.

Step 31 (C): (R)-tert-Butyl methylthio(1-(4-(trifluoromethyl)-phenyl)pyrrolidine-2-carboxamido)methylenecarbamate

A solution of (R)-1-(4-(trifluoromethyl)phenyl)pyrrolidine-2-carboxylic acid from step (B), tert-butyl (4-amino-2,6-dichlorobenzylamino)(amino)-methylenecarbamate (60 mg), Py•BOP (186 mg) and triethylamine (0.15 mL) in dichloromethane (0.20 mL) was stirred at room temperature for 12 h. The crude product was purified by preparative TLC eluting with 50% ethyl acetate/50% hexanes to give the title compound as a colorless oil (24 mg). retention time: 2.31 min (method A). MS (ESI) (M+Na)⁺ 454.12.

Step 31 (D): (R)-tert-Butyl (3,5-dichloro-4-(2-(dimethylamino)acetamido)-benzylamino)(1-(4-(trifluoromethyl)phenyl)-pyrrolidine-2-carboxamido)-methylenecarbamate

A solution of (R)-tert-butyl methylthio(1-(4-(trifluoromethyl)-phenyl)pyrrolidine-2-carboxamido)methylenecarbamate (6 mg), 3,5-dichloro-4-(2-(dimethylamino)acetamido)-benzylamine (10 mg) and triethylamine (50 μL) was heated at 56° C. for 12 h. The crude reaction mixture was purified directly by preparative TLC eluting with 10% methanol/89% dichloromethane/1% ammonia hydroxide to give the title compound as a colorless oil (5 mg). retention time: 2.03 min (method A). MS (ESI) (M+Na)⁺ 659.19.

Step 31 (E): (R,E)-N-(amino(3,5-dichloro-4-(2-(dimethylamino)-acetamido)-benzylamino)methylene)-1-(4-(trifluoromethyl)phenyl)-pyrrolidine-2-carboxamide

To a solution of: (R)-tert-butyl (3,5-dichloro-4-(2-(dimethylamino)-acetamido)benzylamino)(1-(4-(trifluoromethyl)phenyl)-pyrrolidine-2-carboxamido)-methylenecarbamate (4 mg) in dichloromrthane (0.10 mL) was added TFA (0.50 μL), and the resulting mixture was stirred at room temperature for 12 h. The solvents were removed in vacuo to give the title compound as its TFA salt (3 mg). retention time: 1.52 min (method A). MS (ESI) (M+H)⁺ 559.13.

EXAMPLE 32 (2S,4R)—N-((4-Acetamido-3,5-dichlorobenzylamino)(amino)-methylene)-4-methoxy-1-phenylpyrrolidine-2-carboxamide

Step 32 (A): (2S,4R)—N-((4-Acetamido-3,5-dichlorobenzylamino)-(tert-butoxycarbonylamino)methylene)-4-methoxy-1-phenylpyrrolidine-2-carboxamide

Intermediate H and Intermediate B were reacted in the manner of Example 1, step (A), to produce the title compound (93% yield). LC-MS (M+H)⁺=578. LC-MS RT (column 1)=2.548/4.00. ¹H NMR (500 MHz, CDCl₃) δ ppm 12.85 (s, 0.4H), 12.03 (s, 0.6H), 8.82-8.97 (m, 1H), 7.76-7.88 (m, 1H), 7.07-7.33 (m, 4H), 6.72-6.78 (m, 0.4H), 6.44-6.65 (m, 2.6H), 3.97-4.60 (m, 5.4H), 3.65-3.73 (m, 0.6H), 3.26-3.42 (m, 4H), 2.42-2.53 (m, 0.4H), 2.19-2.36 (m, 1.6H), 2.17-2.07 (m, 3H), 1.42 (s, 5.4H), 1.34 (s, 3.6H).

Step 32 (B): The product of step (A) was reacted in the manner of Example 1, step (B), to produce (2S,4R)—N-((4-acetamido-3,5-dichlorobenzylamino)(amino)-methylene)-4-methoxy-1-phenylpyrrolidine-2-carboxamide (72% yield). LC-MS (M+H)⁺=478. LC-MS RT (column 1)=2.018/5.00. ¹H NMR (500 MHz, CD₃OD) δ ppm 7.33 (s, 2H), 7.12 (s, 2H), 6.46-6.68 (m, 3H), 4.07-4.44 (m, 4H), 3.61-3.90 (m, 1H), 3.26-3.42 (m, 4H), 2.28-2.46 (m, 1H), 2.14-2.24 (m, 4H).

EXAMPLE 33 (2S,4R)—N-(Amino(3,5-dichloro-4-(2-(dimethylamino)acetamido)benzylamino)methylene)-4-methoxy-1-phenylpyrrolidine-2-carboxamide

Step 33 (A): (2S,4R)—N-((tert-Butoxycarbonylamino)(3,5-dichloro-4-(2-(dimethylamino)acetamido)benzylamino)methylene)-4-methoxy-1-phenylpyrrolidine-2-carboxamide

Intermediate G and Intermediate F were reacted in the manner of Example 1, step (A) to produce the title compound (78% yield). LC-MS (M+H)⁺=621. LC-MS RT (column 1)=2.293/4.00. ¹H NMR (500 MHz, CDCl₃) δ ppm 12.83 (s, 0.4H), 12.05 (s, 0.6H), 8.81-9.21 (m, 2H), 7.10-7.32 (m, 4H), 6.45-6.79 (m, 3H), 3.99-4.62 (m, 4.4H), 3.67-3.74 (m, 0.6H), 3.28-3.42 (m, 4H), 3.21 (s, 2H), 2.43-2.52 (m, 6.4H), 2.22-2.35 (s, 1.6H), 1.43 (s, 5.4H), 1.34 (s, 3.6H).

Step 33 (B): (2S,4R)—N-(Amino(3,5-dichloro-4-(2-(dimethylamino)acetamido)-benzylamino)methylene)-4-methoxy-1-phenylpyrrolidine-2-carboxamide

The product of step (A) was reacted in the manner of Example 1, step (B), to produce the title compound (84% yield). LC-MS (M+H)⁺=521. LC-MS RT (column 1)=1.502/5.00. ¹H NMR (500 MHz, CD₃OD) δ ppm 7.34 (s, 2H), 7.05-7.21 (s, 2H), 6.42-6.69 (m, 3H), 4.09-4.44 (m, 4H), 3.59-3.90 (m, 1H), 3.26-3.43 (m, 4H), 3.21 (s, 2H), 2.26-2.50 (m, 7H), 2.13-2.26 (m, 1H).

EXAMPLE 34 cis-N-((4-Acetamido-3,5-dichlorobenzylamino)(amino)methylene)-3-phenyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

Step 34 (A): cis-tert-Butyl N-(4-acetamido-3,5-dichlorobenzyl)-N′-(3-phenyl-3-azabicyclo[3.1.0]hexane-2-carbonyl)carbamimidoylcarbamate

The title compound was prepared from Intermediate I and Intermediate B in the same manner as Example 2, step (A) Silica gel chromatography of the crude product eluted with a 0-10% MeOH/CHCl₃ gradient afforded the pure product (80 mg, 87% yield). LC-MS (M+H)⁺=560. LC-MS RT (column 2)=2.152/3.000.

Step 34 (B): cis-N-((4-Acetamido-3,5-dichlorobenzylamino)-(amino)methylene)-3-phenyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

To a solution of the product of step (A) (80 mg, 142 umol) in CH₂Cl₂ (2.0 mL) was added TFA (200 uL). The mixture was stirred at rt for 2.5 h, and then the mixture was washed with saturated aqueous sodium bicarbonate, dried (MgSO₄), filtered, and concentrated in vacuo. The residue was loaded onto a silica gel chromatography column, and eluted with a 0 to 10% MeOH/CHCl₃ gradient to afford the pure product as a white solid (45 mg, 69% yield). LC-MS (M+H)⁺=460. LC-MS RT (column 2)=1.547/3.000. ¹H NMR (500 MHz, CD₃OD) δ ppm 7.41 (s, 2H), 7.10 (s, 2H), 6.63 (s, 1H), 6.48 (s, 2H), 4.38-4.47 (m, 2H), 4.04 (d, J=2.75 Hz, 1H), 3.75 (d, J=7.02 Hz, 1H), 3.31 (s, 1H), 2.16 (s, 3H), 2.01 (br s, 1H), 1.69 (br s, 1H), 0.78 (br s, 1H), 0.57 (br s, 1H)

EXAMPLE 35 cis-N-((3,5-Dichloro-4-(2-(dimethylamino)acetamido)-benzylamino)(amino)methylene)-3-phenyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

Step 35 (A): cis-tert-Butyl N-(3,5-dichloro-4-(2-(dimethylamino)acetamido)-benzyl)-N′-(3-phenyl-3-azabicyclo[3.1.0]hexane-2-carbonyl)carbamimidoylcarbamate

The title compound was prepared from Intermediate I and Intermediate F in the same manner as Example 2, step (A). Silica gel chromatography of the crude product eluted with a 0 to 10% MeOH/CHCl₃ gradient afforded the pure product (76 mg, 77% yield).LC-MS (M+H)⁺=603. LC-MS RT (column 2)=1.940/3.000.

Step 35 (B): cis-N-((3,5-Dichloro-4-(2-(dimethylamino)acetamido)-benzylamino)(amino)methylene)-3-phenyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

The title compound was prepared from the product of step (A) in the same manner as Example 34 to afford the pure product as a white solid (28 mg, 44% yield). LC-MS (M+H)⁺=503. LC-MS RT (column 2)=1.338/3.000. ¹H NMR (500 MHz, CD₃OD) δ ppm 7.43 (s, 2H), 7.10 (t, J=7.17 Hz, 2H), 6.63 (d, J=6.10 Hz, 1H), 6.48 (s, 2H), 4.39-4.48 (m, 2H), 4.04 (d, J=4.27 Hz, 1H), 3.75 (d, J=8.55 Hz, 1H), 3.27-3.30 (m, 1H), 3.20 (s, 2H), 2.40-2.46 (m, 6H), 2.01 (br s, 1H), 1.70 (br s, 1H), 0.79 (br s, 1H), 0.57 (br s, 1H)

Biological Methods

There are a number of methods by which inhibitors of the BACE enzyme can be identified experimentally. The enzyme can be obtained from membrane samples from natural tissues or cultured cells or can be expressed recombinantly in a host cell by well known methods of molecular biology. The whole enzyme or a portion thereof can be expressed, for example, in bacterial, insect or mammalian cells to obtain a catalytically active enzyme species. The enzymatic activity and/or ligand binding capability of the enzyme can be assessed within these membrane samples, or the enzyme can be purified to varying extents. As an illustrative example, the nucleic acid sequence encoding the pro and catalytic domains of human BACE can be appended on the 5′ end with an untranslated and signal sequence from the gene for acetylcholinesterase, and on the 3′ end with a sequence encoding a poly-histidine tag. This cDNA can then be expressed in Drosophila melanogaster S2 cells in which the signal and pro sequences of the transcribed/translated protein are removed by cellular proteases and the catalytic domain, appended by a C-terminal poly-histidine tag, is secreted out into the cellular medium. The enzyme can then be purified from the culture medium by nickel affinity chromatography by methods well known to those trained in the art [Mallender, W. et al., (2001) “Characterization of recombinant, soluble beta-secretase from an insect cell expression system.” Mol. Pharmacol. 59: 619-626]. Similar strategies for expressing and purifying various forms of BACE in bacterial, mammalian and other cell types would be known to one skilled in the art. A preferred method for determining the potency of a test compound in binding to the BACE enzyme is by monitoring the displacement of a suitable radioligand.

Radioligand displacement assays with a radiolabeled BACE inhibitor (WO 2004 013098, compound 3, where the methoxy group is substituted for C(³H)₃) were carried out using standard methods (Keen, M. (1999) in Receptor Binding Techniques (Walker, J. M. ed) p. 106 Humana Press, Totowa, N.J.). The HEK293-9B.A1 cell line, which overexpresses the BACE1 enzyme, was derived from HEK293 cells (Simmons, N. L. (1990) A cultured human renal epithelioid cell line responsive to vasoactive intestinal peptide. Exp. Physiol. 75:309-19.) by RAGE™ (Harrington, J. J. et al. (2001) Creation of genome-wide protein expression libraries using random activation of gene expression. Nat. Biotechnol. 19:440-5.; U.S. Pat. Nos. 6,410,266 and 6,361,972). T225 flask cultures of HEK293-9B.A1 were grown to 80% confluency in DMEM supplemented with 2 mM L-glutamine, 10 μg/ml penicillin, 10 μg/ml streptomycin, 3 μg/ml puromycin, 100 nM methotrexate, and 10% fetal bovine serum (Invitrogen, Carlsbad, Calif.), harvested, and resuspended at 2×10⁸ cells per 10 ml of lysis buffer consisting of 50 mM HEPES pH 7.0 containing a protease inhibitor cocktail of AEBSF 104 μM, aprotinin 80 nM, leupeptin 2 μM, bestatin 4 μM, pepstatin A 1.5 μM, and E-64 1.4 μM (0.1% of protease inhibitor cocktail P8340, Sigma-Aldrich, St. Louis, Mo.) at 4° C. The resuspended cells were homogenized using a Polytron (Brinkman, Westbury, N.Y.) at setting 6 for 10 sec., then centrifuged at 48,000×g for 10 min. The resulting pellet was washed by repeating the resuspension, homogenization and centrifugation steps. The final pellet was resuspended in buffer at 4° C. to yield a total protein concentration of 5 mg/ml, then aliquots were frozen in liquid nitrogen for further storage at −70° C. Immediately before carrying out a binding assay, an aliquot of cell homogenate was thawed and diluted to a concentration of 100 μg/ml in assay buffer consisting of 50 mM HEPES pH 5.0 and 0.1% CHAPSO. Assays were initiated in polypropylene 96-well plates (Costar, Cambridge, Mass.) by the addition of 200 μl of cell homogenate to 50 μl of assay buffer containing 1 nM radioligand (WO 2004 013098, compound 3, where the methoxy group is substituted for C(³H)₃: 80 Ci/mMol) and various concentrations of unlabelled compounds, and incubated for 1.5 hr. at 25° C. Separation of bound from free radioligand was by filtration on GFF glass fiber filters (Innotech Biosystems International, Lansing, Mich.) using an Innotech cell harvester. Filters were washed three times with 0.3 ml of phosphate buffered saline pH 7.0 at 4° C. and assessed for radioactivity using a Wallac 1450 Microbeta liquid scintillation counter (PerkinElmer, Boston, Mass.). Ki values of competing compounds were derived through Cheng-Prussoff correction of IC50 values calculated using XLfit (IDBS, Guildford, UK).

Abbreviations: AEBSF: 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride CHAPSO: 3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate D-MEM: Dulbecco's modified eagle medium HEPES: 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid RAGE™: Random Activation of Gene Expression™

The activity of specific compounds described herein and tested in the above assay is provided in Table 1.

TABLE 1 Compounds of Example Activity Rating 1 ++ 2 ++ 3 ++ 4 ++ 5 ++ 6 ++ 31 +++ 32 +++ 33 +++ 34 ++ ^(a)Activity based on IC₅₀ values: +++ = <0.1 μM ++ = 0.1–1.0 μM + = >1.0 μM

In Vitro Assay to Identify β-Secretase Inhibitor Based on the Inhibition of Aβ Formation from Membrane Preparations.

An isolated membrane fraction which contains functionally active β-secretase and β-APP substrates can generate β-secretase cleavage products including Aβ (Roberts, S. B.; Hendrick, J. P.; Vinitsky, A.; Lewis, M.; Smith, D. W.; Pak, R. PCT Publication WO 01/0175435; Fechteler, K.; Kostka, M.; Fuchs, M. Patent Application No. DE 99-19941039; Shearman, M.; Beher, D. et al., Biochemistry, 2000, 39, 8698-8704; Zhang, L. Song, L. et al., Biochemistry 2001, 40, 5049-5055). An isolated membrane fraction can be prepared from human derived cell lines such as HeLa and H4 which have been transfected with wild type or mutant forms of β-APP or a human alkaline phosphatase β-APP fusion construct, and stably express high levels of β-secretase substrates. The endogenous β-secretase present in the isolated membranes prepared at 0-4° C. cleaves the β-APP substrates when the membranes are shifted from 0-4 to 37° C. Detection of the cleavage products including Aβ can be monitored by standard techniques such as immunoprecipitation (Citron, M.; Diehl, T. S. et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 13170-13175), western blot (Klafki, H.-W.; Ambramowski, D. et al., J. Biol. Chem. 1996, 271, 28655-28659), enzyme linked immunosorbent assay (ELISA) as demonstrated by Seubert, P.; Vigo-Pelfrey, C. et al., Nature, 1992, 359, 325-327, or by a preferred method using time-resolved fluorescence of the homogeneous sample containing membranes and Aβ (Roberts, S. B.; Hendrick, J. P.; Vinitsky, A.; Lewis, M.; Smith, D. W.; Pak, R. PCT Publication WO 01/0175435; Shearman, M.; Beher, D. et al., Biochemistry, 2000, 39, 8698-8704). The Aβ present in a homogeneous sample containing membranes can be detected by time-resolved fluorescence with two antibodies that recognize different epitopes of Aβ. One of the antibodies recognizes an epitope that is present in Aβ but not present in the precursor fragments; preferably the antibody binds the carboxyl terminus of Aβ generated by the β-secretase cleavage. The second antibody binds to any other epitope present on Aβ. For example, antibodies that bind the N-terminal region (e.g., 26D6-B2-B3′ SIBIA Neurosciences, La Jolla, Calif.) or bind the C-terminal end (e.g., 9S3.2® antibody, Biosolutions, Newark, Del.) of the Aβ peptide are known. The antibodies are labeled with a pair of fluorescent adducts that transfer fluorescent energy when the adducts are brought in close proximity as a result of binding to the N- and C-terminal ends or regions of Aβ. A lack of fluorescence is indicative of the absence of cleavage products, resulting from inhibition of β-secretase. The isolated membrane assay can be used to identify candidate agents that inhibit the activity of β-secretase cleavage and Aβ production.

A typical membrane-based assay requires 45 μg membrane protein per well in a 96- or 384-well format. Membranes in a neutral buffer are combined with the test compound and shifted from 0-4 to 37° C. Test agents may typically consist of synthetic compounds, secondary metabolites from bacterial or fungal fermentation extracts, or extracts from plant or marine samples. All synthetic agents are initially screened at doses ranging from 10-100 μM or in the case of extracts at sufficient dilution to minimize cytotoxicity. Incubation of the membranes with the test agent will continue for approximately 90 minutes at which time fluorescence labeled antibodies are added to each well for Aβ quantitation. The time-resolved fluorescence detection and quantitation of Aβ is described elsewhere (Roberts, S. B.; Hendrick, J. P.; Vinitsky, A.; Lewis, M.; Smith, D. W.; Pak, R. PCT Publication WO 01/0175435; Shearman, M.; Beher, D. et al., Biochemistry, 2000. 39, 8698-8704). Results are obtained by analysis of the plate in a fluorescence plate reader and comparison to the mock treated membranes and samples in which known amounts of Aβ were added to construct a standard concentration curve. A positive acting compound is one that inhibits the Aβ relative to the control sample by at least 50% at the initial tested concentration. Compounds of the present application are considered active when tested in the above assay if the IC₅₀ value for the test compound is less than 50 μM. A preferred IC₅₀ value is less than 1 μM. A more preferred IC₅₀ value is less than 0.1 μM. If a compound is found to be active then a dose response experiment is performed to determine the lowest dose of compound necessary to elicit the inhibition of the production of Aβ.

In Vivo Assays for the Determination of Aβ Reduction by a β-Secretase Inhibitor.

In vivo assays are available to demonstrate the inhibition of β-secretase activity. In these assays, animals, such as mice, that express normal levels of APP, β- and γ-secretase or are engineered to express higher levels of APP and hence Aβ can be used to demonstrate the utility of β-secretase inhibitors, as demonstrated with γ-secretase inhibitors [Dovey, H. et al., (2001), J. Neurochem. 76: 173-181]. In these assays, β-secretase inhibitors are administered to animals and Aβ levels in multiple compartments, such as plasma, cerebral spinal fluid, and brain extracts, are monitored for Aβ levels using methods previously outlined. For instance, Tg2576 mice, which overexpress human APP, are administered β-secretase inhibitors by oral gavage at doses that will cause measurable Aβ lowering, typically less than 100 mg/kg. Three hours after dosing plasma, brain, and CSF are collected, frozen in liquid nitrogen, and stored at −80° C. until analysis. For Aβ detection, plasma is diluted 15-fold in PBS with 0.1% Chaps while CSF is diluted 15-fold in 1% Chaps with protease inhibitors (5 μg/ml leupeptin, 30 μg/ml aprotinin, 1 mM phenylmethylsulfonylfluoride, 1 μM pepstatin). Brains are homogenized in 1% Chaps with protease inhibitors using 24 ml solution/g brain tissue. Homogenates were then centrifuged at 100,000×g for 1 hr at 4° C. The resulting supernatants were then diluted 10-fold in 1% Chaps with protease inhibitors. Aβ levels in the plasma, CSF, and brain lysate can then be measured using time-resolved fluorescence of the homogenous sample or one of the other methods previously described.

A β-secretase inhibitor is considered active in one of the above in vivo assays if it reduces Aβ by at least 50% at a dosage of 100 mg/kg.

Dosage and Formulation

The compounds of the present application can be administered orally using any pharmaceutically acceptable dosage form known in the art for such administration. The active ingredient can be supplied in solid dosage forms such as dry powders, granules, tablets or capsules, or in liquid dosage forms, such as syrups or aqueous suspensions. The active ingredient can be administered alone, but is generally administered with a pharmaceutical carrier. A valuable treatise with respect to pharmaceutical dosage forms is Remington's Pharmaceutical Sciences, Mack Publishing.

The compounds of the present application can be administered in such oral dosage forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. Likewise, they may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be employed to prevent or treat neurological disorders related to β-amyloid production or accumulation, such as Alzheimer's disease and Down's Syndrome.

The compounds of this application can be administered by any means that produces contact of the active agent with the agent's site of action in the body of a host, such as a human or a mammal. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

The dosage regimen for the compounds of the present application will, of course, vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.

Advantageously, compounds of the present application may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.

The compounds for the present application can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

In the methods of the present application, the compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients, or carriers (collectively referred to herein as carrier materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl callulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like; for oral administration in liquid form, the oral drug components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or β-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

The compounds of the present disclosure can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamallar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.

Compounds of the present disclosure may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds of the present disclosure may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.

Gelatin capsules may contain the active ingredient and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.

Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. 

1. A compound of Formula (I); or a stereoisomer thereof

wherein R₁ is phenyl optionally substituted with one or more groups selected from halogen, CN, CF₃, OH, —NH₂, C₃₋₆cycloalkyl, C₁₋₆alkoxy, C₂₋₆alkenyl, C₁₋₆alkyl optionally substituted with OH, C₃₋₆cycloalkyl, or —NH₂, —(CH₂)_(m)—NHC(═O)OC₁₋₆ alkyl, —(CH₂)_(m)—NHC(═O)Ophenyl optionally substituted with halogen, —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉; R₂ and R₃ are each independently hydrogen, methyl or hydroxymethyl; p is 0 or 1; m is 0 or 1; R₄ and R₆ are independently hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy, phenyl or CF₃ in which said phenyl is optionally substituted with one or more groups selected from halogen, CN, CF₃ and OCH₃; R₅ and R₅ are each independently hydrogen, C₁₋₆alkyl, or C₁₋₄alkanol; R₇ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ is C₁₋₆alkyl or C₃₋₆cycloalkyl in which each is optionally substituted with a group selected from halogen, CN, CF₃ and C₁₋₄alkoxy; R₉ is —C₁₋₆alkylNR₁₀R₁₁; R₁₀ is hydrogen or C₁₋₆alkyl; and R₁₁ is hydrogen, C₁₋₆alkyl optionally substituted with OH, halogen, C₁₋₄alkoxy, C₃₋₆cycloalkyl, thienyl or C₁₋₄alkylcarboxyl, —(CH₂)_(m)C₃₋₆cycloalkyl optionally substituted with phenyl or C₁₋₄alkyl; —(CH₂)_(m)phenyl optionally substituted with halogen, hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy; or R₁₀ and R₁₁ together with the nitrogen to which they are attached is azetidine, aziridine, pyrrolidine, piperidine, homopiperidine, homopiperazine, morpholine or thiomorpholine, in which each is optionally substituted with a group selected from halogen, C₁₋₆alkyl and C₁₋₄alkoxy; or a nontoxic pharmaceutically acceptable salt thereof.
 2. A compound of Formula (II); or a stereoisomer thereof

wherein R₁ is phenyl optionally substituted with one or more groups selected from halogen, CN, CF₃, OH, —NH₂, C₃₋₆cycloalkyl, C₁₋₆alkoxy, C₂₋₆alkenyl, C₁₋₆alkyl optionally substituted with OH, C₃₋₆cycloalkyl, or —NH₂, —(CH₂)_(m)—NHC(═O)OC₁₋₆ alkyl, —(CH₂)_(m)—NHC(═O)Ophenyl optionally substituted with halogen, —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉; p is 0 or 1; m is 0 or 1; R₄ is hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy, phenyl or CF₃ in which said phenyl is optionally substituted with one or more groups selected from halogen, CN, CF₃ and OCH₃; R₇ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ is C₁₋₆alkyl or C₃₋₆cycloalkyl in which each is optionally substituted with a group selected from halogen, CN, CF₃ and C₁₋₄alkoxy; R₉ is —C₁₋₆alkylNR₁₀R₁₁; R₁₀ is hydrogen or C₁₋₆alkyl; and R₁₁ is hydrogen, C₁₋₆alkyl optionally substituted with OH, halogen, C₁₋₄alkoxy, C₃₋₆cycloalkyl, thienyl or C₁₋₄alkylcarboxyl, —(CH₂)_(m)C₃₋₆cycloalkyl optionally substituted with phenyl or C₁₋₄alkyl; —(CH₂)_(m)phenyl optionally substituted with halogen, hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy; or R₁₀ and R₁₁ together with the nitrogen to which they are attached is azetidine, aziridine, pyrrolidine, piperidine, homopiperidine, homopiperazine, morpholine or thiomorpholine, in which each is optionally substituted with a group selected from halogen, C₁₋₆alkyl and C₁₋₄alkoxy; or a nontoxic pharmaceutically acceptable salt thereof.
 3. A compound of Formula (III); or a stereoisomer thereof

wherein R₁ is phenyl optionally substituted with one or more groups selected from halogen, C₃₋₆cycloalkyl, C₁₋₆alkyl optionally substituted with cyclopropyl, or —(CH₂)_(m)—, —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉; R₄ is hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy, phenyl or CF₃ in which said phenyl is optionally substituted with one or more groups selected from halogen, CN, CF₃ and OCH₃; R₇ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ is C₁₋₆alkyl or C₃₋₆cycloalkyl in which each is optionally substituted with a group selected from halogen, CN, CF₃ and C₁₋₄alkoxy; R₉ is —C₁₋₆alkylNR₁₀R₁₁; R₁₀ is hydrogen or C₁₋₆alkyl; and R₁₁ is hydrogen, C₁₋₆alkyl optionally substituted with OH, halogen, C₁₋₄alkoxy, C₃₋₆cycloalkyl, thienyl or C₁₋₄alkylcarboxyl, —(CH₂)_(m)C₃₋₆cycloalkyl optionally substituted with phenyl or C₁₋₄alkyl; —(CH₂)_(m)phenyl optionally substituted with halogen, hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy; or R₁₀ and R₁₁ together with the nitrogen to which they are attached is azetidine, aziridine, pyrrolidine, piperidine, homopiperidine, homopiperazine, morpholine or thiomorpholine, in which each is optionally substituted with a group selected from halogen, C₁₋₆alkyl and C₁₋₄alkoxy; m is 0 or 1; or a nontoxic pharmaceutically acceptable salt thereof.
 4. A compound of Formula (IV);

wherein R₁ is phenyl optionally substituted with one or more groups selected from halogen, C₃₋₆cycloalkyl, C₁₋₆alkyl optionally substituted with cyclopropyl, or —(CH₂)_(m)—, —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉; R₄ is hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy, phenyl or CF₃ in which said phenyl is optionally substituted with one or more groups selected from halogen, CN, CF₃ and OCH₃; R₇ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ is C₁₋₆alkyl or C₃₋₆cycloalkyl in which each is optionally substituted with a group selected from halogen, CN, CF₃ and C₁₋₄alkoxy; R₉ is —C₁₋₆alkylNR₁₀R₁₁; R₁₀ is hydrogen or C₁₋₆alkyl; and R₁₁ is hydrogen, C₁₋₆alkyl optionally substituted with OH, halogen, C₁₋₄alkoxy, C₃₋₆cycloalkyl, thienyl or C₁₋₄alkylcarboxyl, —(CH₂)_(m)C₃₋₆cycloalkyl optionally substituted with phenyl or C₁₋₄alkyl; —(CH₂)_(m)phenyl optionally substituted with halogen, hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy; or R₁₀ and R₁₁ together with the nitrogen to which they are attached is azetidine, aziridine, pyrrolidine, piperidine, homopiperidine, homopiperazine, morpholine or thiomorpholine, in which each is optionally substituted with a group selected from halogen, C₁₋₆alkyl and C₁₋₄alkoxy; m is 0 or 1; or a nontoxic pharmaceutically acceptable salt thereof.
 5. A compound of Formula (IV);

wherein R₁ is phenyl optionally substituted with one or more groups selected from halogen, C₃₋₆cycloalkyl, C₁₋₆alkyl optionally substituted with cyclopropyl, or —(CH₂)_(m)—, —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉; R₄ is hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, benzyloxy, phenyl or CF₃ in which said phenyl is optionally substituted with one or more groups selected from halogen, CN, CF₃ and OCH₃; R₇ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ is C₁₋₆alkyl or C₃₋₆cycloalkyl in which each is optionally substituted with a group selected from halogen, CN, CF₃ and C₁₋₄alkoxy; R₉ is —C₁₋₆alkylNR₁₀R₁₁; R₁₀ is hydrogen or C₁₋₆alkyl; and R₁₁ is hydrogen, C₁₋₆alkyl optionally substituted with OH, halogen, C₁₋₄alkoxy, C₃₋₆cycloalkyl, —(CH₂)_(m)C₃₋₆cycloalkyl; —(CH₂)_(m)phenyl optionally substituted with halogen, hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy; or R₁₀ and R₁₁ together with the nitrogen to which they are attached is azetidine, aziridine, pyrrolidine, piperidine, homopiperidine, homopiperazine, morpholine or thiomorpholine; m is 0 or 1; or a nontoxic pharmaceutically acceptable salt thereof.
 6. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1 in association with a pharmaceutically acceptable adjuvant, carrier or diluent.
 7. A method for the treatment of Alzheimer's Disease, cerebral amyloid angiopathy and Down's Syndrome which comprises administering to a mammal in need thereof a therapeutically effective amount of a compound of claim
 1. 